Immunotherapy for clearing pathological tau conformers

ABSTRACT

The present invention relates to methods of treating and preventing Alzheimer&#39;s Disease or other tauopathies in a subject by administering a tau protein, its immunogenic epitopes, or antibodies recognizing the tau protein or its immunogenic epitopes under conditions effective to treat or prevent Alzheimer&#39;s Disease of other tauopathies. Also disclosed are methods of promoting clearance of aggregates from the brain of the subject and of slowing progression of tangle-related behavioral phenotype in a subject.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/787,051, filed Mar. 29, 2006, which is herebyincorporated by reference.

The subject matter of this application was made with support from theUnited States Government under NIH/NIA, Grant No. AG20197. The U.S.Government may have certain rights.

FIELD OF THE INVENTION

The present invention is directed to a method of preventing and treatingAlzheimer's disease and inhibiting accumulation of tau neurofibrillarytangles in a subject.

BACKGROUND OF THE INVENTION

An emerging treatment for Alzheimer's disease (AD) is immunotherapy toclear amyloid-β (Aβ). Another important target in AD and frontotemporaldementia is the neurofibrillary tangles and/or their pathological tauprotein conformers, whose presence correlates well with the degree ofdementia (Terry, R., “Neuropathological Changes in Alzheimer Disease,”Prog Brain Res. 101:383-390 (1994); Goedert, M., “Tau Protein andNeurodegeneration,” Semin Cell Dev Biol. 15:45-49 (2004)). The objectiveof immunotherapy for tau pathology is that anti-tau antibodies can cleartau aggregates that may affect neuronal viability. Tau is a solubleprotein that promotes tubulun assembly, microtubule stability, andcytoskeletal integrity. Although tau pathology is likely to occurfollowing Aβ aggregation based on Down syndrome studies, analyses of ADbrains and mouse models indicate that these pathologies are likely to besynergistic (Sigurdsson, et al., “Local and Distant HistopathologicalEffects of Unilateral Amyloid-beta 25-35 Injections into the Amygdala ofYog F344 Rats,” Neurobiol Aging 17:893-901(1996); Sigurdsson, et al.,“Bilateral Injections of Amyloid-β 25-35 into the Amygdala of YoungFischer Rats: Behavioral, Neurochemical, and Time DependentHistopathological Effects,” Neurobiol Aging 18:591-608 (1997); Lewis, etal., “Neurofibrillary Tangles, Amyotrophy and Progressive MotorDisturbance in Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet.25:402-405 (2000); Gotz, et al., “Formation of Neurofibrillary Tanglesin P301L Tau Transgenic Mice Induced by A-beta 42 Fibrils,” Science293:1491-1495 (2001); Delacourte, et al., “Nonoverlapping but SynergeticTau and APP Pathologies in Sporadic Alzheimer's Disease,” Neurology.59:398-407 (2002); Oddo, et al., “Abeta Inmunotherapy Leads to Clearanceof Early, But Not Late, Hyperphosphorylated Tau Aggregates via theProteasome,” Neuron 43:321-332 (2004); Ribe, et al., “AcceleratedAmyloid Deposition, Neurofibrillary Degeneration and Neuronal Loss inDouble Mutant APP/Tau Transgenic Mice,” Neurobiol Dis. (2005)). Hence,targeting both pathologies may substantially increase treatmentefficacy. To date, no tau mutations have been observed in AD, however,in frontotemporal dementia, mutations in the tau protein on chromosome17 (FTDP-17) are a causative factor in the disease, which furthersupports tau-based therapeutic approaches (Poorkaj, et al., “Tau is aCandidate Gene for Chromosome 17 Frontotemporal Dementia,” Ann Neurol.43:815-825 (1998); Spillantini, et al., “Frontotemporal Dementia andParkinsonism Linked to Chromosome 17: A New Group of Tauopathies,” BrainPathol. 8:387-402 (1998)). Transgenic mice expressing these mutationshave modeled many aspects of the disease and are valuable tools to studythe pathogenesis of tangle-related neurodegeneration and to assesspotential therapies. One of these models, the P301L mouse model (Lewis,et al., “Neurofibrillary Tangles, Amyotrophy and Progressive MotorDisturbance in Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet.25:402-405 (2000)), recapitulates many of the features of frontotemporaldementia although the CNS distribution of the tau aggregates resultsprimarily in sensorimotor abnormalities which complicates cognitiveassessment. Homozygous lines of this mouse model have an early onset ofCNS pathology and associated functional impairments which make themideal for the initial assessment of the feasibility of immunotherapy,targeting pathological tau conformers.

Other tau-related therapeutic approaches include: (1) drugs that inhibitthe kinases or activate the phosphatases that affect the state of tauphosphorylation (Iqbal, et al. “Inhibition of NeurofibrillaryDegeneration: A Promising Approach to Alzheimer's Disease and OtherTauopathies,” Curr Drug Targets 5:495-502 (2004); Noble, et al.,Inhibition of Glycogen Synthase Kinase-3 by Lithium Correlates withReduced Tauopathy and Degeneration In Vivo,” Proc Natl Acad Sci USA102:6990-6995 (2005)); (2) microtubule stabilizing drugs (Michaelis, etal., {beta}-Amyloid-Induced Neurodegeneration and Protection byStructurally Diverse Microtubule-Stabilizing Agents,” J Pharmacol ExpTher. 312:659-668 (2005); Zhang, et al., “Microtubule-Binding DrugsOffset Tau Sequestration by Stabilizing Microtubules and Reversing FastAxonal Transport Deficits in a Tauopathy Model,” Proc Natl Acad Sci USA102:227-231 (2005)); (3) compounds that interfere with tau aggregation(Pickhardt, et al., “Anthraquinones Inhibit Tau Aggregation and DissolveAlzheimer's Paired Helical Filaments In Vitro and in Cells,” J BiolChem. 280:3628-3635 (2005)); and (4) drugs that promote heat shockprotein mediated clearance of tau (Dickey, et al., “Development of aHigh Throughput Drug Screening Assay for the Detection of Changes in TauLevels—Proof of Concept with HSP90 Inhibitors,˜ Curr Alzheimer Res.2:231-238 (2005)). While all these approaches are certainly worthpursuing, target specificity and toxicity are of a concern, whichemphasizes the importance of concurrently developing other types oftau-targeting treatments, such as immunotherapy.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention includes a method of preventing ortreating Alzheimer's Disease or other tauopathies in a subject. Themethod includes administering a tau protein, its immunogenic epitopes,or antibodies recognizing the tau protein or its immunogenic epitopesunder conditions effective to prevent or treat Alzheimer's Disease orother tauopathies.

Another aspect of the present invention includes a method of promotingclearance of aggregates from the brain of a subject. This methodincludes administering a tau protein, its immunogenic epitopes, orantibodies recognizing the tau protein or its immunogenic epitopes underconditions effective to promote clearance of aggregates from the brainof a subject.

A third aspect of the present invention includes a method of slowingprogression of a tangle-related behavioral phenotype in a subject. Thismethod includes administering a tau protein, its immunogenic epitopes,or antibodies recognizing the tau protein or its immunogenic epitopesunder conditions effective to slow a tangle-related behavioral phenotypein a subject.

A fourth aspect of the present invention includes a peptide comprisingan immunogenic epitope of a tau protein- The amino acid sequence of thepeptide can be any one of SEQ ID NOs: 1-20. The immunogenic epitope iseffective in preventing and treating Alzheimer's Disease or othertauopathies in a subject, promoting the clearance of aggregates from thebrain of a subject, and slowing the progression of a tangle-relatedbehavioral phenotype in a subject.

It is hypothesized that clearance of extracellular tangles may reduceassociated pathology, and numerous reports of neuronal uptake ofantibodies suggest that intracellular tangles and pre-tangles may alsobe affected (Fabian, et al., “Intraneuronal IgG in the Central NervousSystem” J Neurol Sci. 73:257-267 (1986); Fabian, et al., “IntraneuronalIgG in the Central Nervous System: Uptake by Retrograde AxonalTransport,” Neurology 37:1780-1784 (1987); Liu, et al.,“Immunohistochemical Localization of Intracellular Plasma Proteins inthe Human Central Nervous System,” Acta Neuropathol (Berl) 78:16-21(1989); Dietzschold, et al., “Delineation of Putative MechanismsInvolved in Antibody-Mediated Clearance of Rabies Virus from the CentralNervous System,” Proc Natl Acad Sci USA 89:7252-7256 (1992) (publishederratum appears in Proc Natl Acad Sci USA 89(19):9365 (1992)); Aihara,et al., “Immunocytochemical Localization of Immunoglobulins in the RatBrain: Relationship to the Blood-Brain Barrier,” J Comp Neurol.342:481-496 (1994); Mohamed, et al., “Immunoglobulin Fc Gamma ReceptorPromotes Immunoglobulin Uptake, Immunoglobulin-Mediated CalciumIncrease, and Neurotransmitter Release in Motor Neurons,” J NeurosciRes. 69:110-116 (2002), which are hereby incorporated by reference intheir entirety). In the present invention, the effectiveness of activeimmunization directed against phosphorylated tau conformers in the CNSwas determined. Towards this end, homozygous P301L mice were immunizedwith a phosphorylated tau epitope with subsequent analysis of taupathology and associated functional impairments. While these studieswere underway, the feasibility of this approach was strengthened byfindings, indicating that vaccination with recombinant x-synuclein intransgenic mice reduces intraneuronal α-synuclein aggregates (Masliah,et al., “Effects of Alpha-Synuclein Immunization in a Mouse Model ofParkinson's Disease,” Neuron 46:857-868 (2005), which is herebyincorporated by reference in its entirety)

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show that phospho-tau-derivative peptide is notfibrillogenic. It is highly immunogenic in mice treated from 2 to 5months of age, but autoantibodies against tau are detected. Homozygoustransgenic (Tg) P301L mice were immunized from 2 months of age with aphosphorylated tau peptide (Phos-tau=Tau379-408[P-Ser_(396,404)]; n=12).Control Tg P301L animals received aluminum adjuvant alone (n=13). Plasmasamples from the animals were analyzed by ELISA, and the brains wereanalyzed biochemically and immunohistochemically at 5 months of age.FIG. 1A shows that the tau derivative does not form fibrils compared toAβI-42 that is very fibrillogenic as depicted by a Thioflavin T assay.FIG. 1B shows the generation of IgG antibodies (1:200 plasma dilution)against the immunogen at various time points (T0, T1, T2, T3=0, 6, 10,and 14 weeks) as determined by phos-tau peptide ELISA assay. Controlmice had low levels of autoantibodies that recognized the immunogen andthose increased with age. FIG. 1C shows that autoantibodies whichrecognize both P301L and wild-type human tau were observed both incontrols and immunized mice but the levels did not differ significantlybetween the groups.

FIGS. 2A-E show that the vaccine reduces tau aggregates in the brains ofP301L tangle mice at 5 months of age. In FIG. 2A, quantitative analysisof MC1 immunoreactivity within the granular layer of the dentate gyrusrevealed a 74% reduction (**p<0.01) in immunized mice compared tocontrol Tg mice that received adjuvant alone. Likewise, in FIG. 2B, PHF1immunoreactivity within the granular layer of the dentate gyrus wasreduced by 52% (*p<0.05) in immunized mice compared to controls. In FIG.2C, further confirmation of a therapeutic effect was obtained byanalysis of the motor cortex, in which MC1 neuronal staining was reducedby 96% (***p<0.0001) compared to control Tg mice. Likewise, in FIG. 2D,in the brainstem, MC1 neuronal staining was reduced by 93% (**p=0.01)compared to control Tg mice. FIG. 2E shows the densitometric analysis ofPHF1 blots. This analysis revealed a strong trend for reduction ininsoluble tau (28% reduction, p-0.09) and a significant increase insoluble tau (77% increase, p=0.01) in the immunized mice compared tocontrol Tg mice, relative to total tau levels. Further analysis of theratio of soluble tau to insoluble tau indicated a significant increasein the immunized group on PHF1 blots (89% increase, p=0.01), suggestinga mobilization of tau from its insoluble form to soluble form in thesetreated animals. The right panel of FIG. 2E shows representative blotsfrom control- and Phos-tau immunized mice. The PHF1 antibody recognizesphosphorylated serines 396 and 404 located within themicrotubule-binding repeat on the C-terminal of paired helical fragment(PHF) tau protein. An antibody against total tau (3G6) was used as acontrol. The same amount of protein was loaded in each lane. Mean valuesare presented with standard error of the mean.

FIGS. 3A-H show that the immunotherapy reduces pathological tau inneurons. Representative examples of the histological regions that wereanalyzed in MC1- and PHF1-stained brain sections are shown. Neuronal tauaggregates were cleared in the dentate gyrus (DG), the motor cortex(MCx), and the brain stem (BS) in immunized mice compared to controlmice. The dentate gyrus develops extensive tau pathology at an early agein the homozygous P301L mice and tau pathology in the motor cortex andbrain stem may relate to the motor deficits in this model. FIGS. 3A and3B show MC1-stained coronal sections through the dentate gyrus incontrol (FIG. 3A) vs. immunized (FIG. 3B) Tg mouse (originalmagnification: 200×). FIG. 3C and 3D show PHF1-stained coronal sectionsthrough the dentate gyrus in a control (FIG. 3C) vs. immunized (FIG. 3D)Tg mouse (original magnification: 200×). FIGS. 3E and 3F showMC1-stained coronal sections through the motor cortex in a control (FIG.3E) vs. immunized (FIG. 3F) Tg mouse (original magnification: 100×).FIGS. 3G and 3H show MC1-stained coronal sections through the brain stembelow the aqueduct of Sylvius in a control (FIG. 3G) vs. immunized (FIG.3H) Tg mouse (original magnification: 100×). These magnifications wereused for the quantitative analysis (Dentate gyrus: 200×: Motor cortex:100×, Brain Stem: 100×).

FIGS. 4A-B show that in mice treated from 2 to 8 months of age, theimmunogenicity of the vaccine is confirmed but high levels ofautoantibodies are detected. Homozygous transgenic (Tg) P301L mice wereimmunized from 2 months of age with phospho tau peptide(Tau379-408[P-Ser_(396,404)]; n=12). Control Tg P301L animals receivedaluminum adjuvant alone (n=12). Sensorimotor performance was assessed at5- and 8 months of age. FIG. 4A shows the generation of antibodies(1:200 plasma dilution) against the immunogen at various time points(T0, T1, T2, T3, T4=0, 4, 8, 14, and 26 weeks) as determined by phosphotau peptide ELISA assay. FIG. 4B shows that autoantibodies thatrecognize both P301L and wild-type human tau were observed in controlsand immunized mice, but the levels did not differ significantly betweenthe groups.

FIGS. 5A-D show that immunotherapy from 2 to 8 months of age slows theprogression of behavioral abnormalities in P301L mice. FIG. 5A shows theperformance of Tg P301L immunized and Tg control mice, trained to remainon a rotarod, and the speed attained during the task. The immunizationincreased the time the animals were able to stay on the rotarod both at5 months (trials 1-3, p<0.02) and 8 months (trials 4-6, p<0.05). FIG. 5Bshows the number of foot slips the animals had during the traverse beamtask. The immunization greatly reduced the number of foot slips duringthe performance of the task at 5 months (p<0.001) and at 8 monthsp=0.05). As set forth in FIG. 5C, there was an increase in the maximumvelocity (Vmax) attained by the phospho tau immunized Tg animals(p=0.004) at 5 months, compared to Tg controls. Vmax did not differbetween the groups at 8 months There was no significant difference inthe distance traveled, average speed (Vmean) or the resting time at 5and 8 months. As shown in FIG. 5D, no difference was observed betweenthe groups in the Object Recognition Task that measures short termmemory. Both the immunized P301L mice and their transgenic controlsspent a comparable time exploring the novel object that differedsubstantially from the time they spent with the old object. This findingindicates that both groups had normal short term memory at 8 months ofage.

FIGS. 6A-D demonstrate that immunotherapy from 2 to 8 months reducesbrain tau pathology. As shown in FIG. 6A, quantitative analysis of MC1immunoreactivity within the granular layer of the dentate gyrus revealeda 47% reduction (*p<0.05) in immunized mice compared to control Tg micethat received adjuvant alone. As shown in FIG. 6B, there was a strongtrend for a diminished PHF1 immunoreactivity within the granular layerof the dentate gyrus (40% reduction; p<0.12) in immunized mice comparedto controls. FIG. 6C shows that as at 5 months of age (see FIG. 3E-F),MC1 neuronal staining of the motor cortex revealed a more pronouncedtherapeutic effect (76%, p=0.02) than in the dentate gyrus. Likewise, asdepicted in FIG. 6D, neuronal MC1 staining was substantially reduced inthe brain stem of immunized mice compared to controls (78%, p=0.005,FIG. 6D). Mean values are presented with standard error of the mean.

FIGS. 7A-L show that purified antibodies from immunized mice stain tauaggregates/tangles in neuronal cell bodies in P301L mice similar to thePHF1 antibody. Adjacent coronal brain sections are depicted through thedentate gyrus (FIGS. 7A-D), motor cortex (FIGS. 7E-H), and brain stem(FIGS. 7I-L), immediately below the Aqueduct of Sylvius in a P301Ltransgenic mouse with tau pathology. In FIG. 7A, the PHF1 antibodyreveals the typical staining of tau aggregates/tangles in neuronal cellbodies as previously reported in this model (Lewis, et al.,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet. 25:402-405(2000), which is hereby incorporated by reference in its entirety). Asshown in FIG. 7C, antibodies from mice immunized with the Phos-taupeptide, which contains the PHF1 epitope, stain primarily neuronal cellbodies within the dentate gyrus and the pattern is similar although notidentical to the PHF1 staining. A similar staining pattern as in FIGS.7A and 7C was observed in the motor cortex (FIGS. 7E and 7G) and brainstem (FIGS. 7I and 7K) following immunoreactivity with the PHF1 antibodyand the polyclonal antibodies from an immunized mouse, respectively.However, this particular polyclonal antibody stained neurons in thebrain stem less intensely than in the dentate gyrus and motor cortex.FIGS. 7B, 7D, 7F, 7H, 7J, and 7L depict adjacent coronal sections tothose shown in FIG. 7A, 7C, 7E, 7G, 7I, and 7K, that were stained withpurified antibodies from Tg control mice that received adjuvant alone(control IgG) or pooled mouse IgG [wild-type (Wt) IgG (Sigma)]. Stainingwith those antibodies resulted in minimal or no staining. Additionally,no immunostaining was observed in wild-type mice with the antibodiespurified from immunized mice. These findings indicate that the immunizedmice generate antibodies that specifically recognize pathological tauaggregates in the P301L mouse. Staining was performed as detailed infrawith PHF1. and purified IgG used at a 1:250 and 10 μg/ml dilution,respectively. Original magnification: 400×.

FIGS. 8A-C show purified antibodies from immunized mice stain tauaggregates/tangles in neuronal cell bodies in Alzheimer's diseasesimilar to the PHF1 antibody. FIG. 5A shows that PHF1 staining of theentorhinal cortex from an Alzheimer's brain reveals the typical stainingof cell bodies and dystrophic neurites as previously described for thisantibody. In FIG. 8B, the polyclonal IgG antibodies derived from animmunized mouse stain neuronal cell bodies in a similar manner as thePHF1 antibody but dystrophic neurites are not prominent. FIG. 8C showsthat antibodies purified from a control mouse that received adjuvantalone do not result in appreciable staining. Overall, the stainingpattern with these different antibodies is comparable to that observedin the P301L mouse (see FIG. 7).

FIGS. 9A-B show intracerebral antibodies that label neurons are detectedin the immunized P301L mice. FIG. 9A shows a coronal brain sectionstained with an anti-IgG secondary antibody (1:50, Vectastain Elite Kit)through the dentate gyrus of the hippocampus of a Phos-tau immunizedP301L mouse. Note the staining of neuronal cell bodies (arrows) andprocesses (arrows) indicating the presence of IgG. No immunostaining isobserved in a non-immunized P301L of a similar age (FIG. 9B) or in awild-type mouse under these staining conditions. Original magnification:400×.

FIGS. 10 shows a coronal brain section through the brachium of theinferior colliculus revealing FITC labeled neurons (arrows).Counterstain with DAPI (blue) shows nuclei of the neurons. Some FITClabeling was observed in a control P310L mouse of the same age that wasinjected with tagged antibodies from a control Tg mouse but neurons werenot detected. No appreciable FITC fluorescence is observed in awild-type mouse of the same age that received intracarotid injection ofantibodies from an immunized mouse or a control mouse.

FIGS. 11A-F show neurons that label with the injected FITC-taggedantibody from an immunized mouse stain with MC1 and PHF1 antibodies. InFIGS. 11A-C, a coronal brain section through the pyramidal layer of thehippocampus are shown. Note the FITC-labeled neurons in FIG. 11A thatstain with PHF1 antibody that was applied to the section (FIG. 11B;Texas red tagged secondary antibody). The section was counterstainedwith DAPI that stains nuclei in blue and double labeled neurons areorange (FIG. 11C). Original magnification: 200×. FIG. 11D-F show acoronal brain section through the nucleus of the brachium inferiorcolliculus (BIC). Note the FITC-labeled neurons in FIG. 11A that stainwith MC1 antibody that was applied to the section (FIG. 11B; Texas redtagged secondary antibody). The section was counterstained with DAPIthat stains nuclei in blue and double labeled neurons are orange (FIG.11C). Original magnification: 200×. For clarification, the labeledneurons in the center of each panel are shown magnified in the insertedboxes.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention includes a method of treating orpreventing Alzheimer's Disease or other tauopathies in a subject. Thismethod includes administering a tau protein, its immunogenic epitopes,or antibodies recognizing the tau protein or its immunogenic epitopesunder conditions effective to treat or prevent Alzheimer's Disease orother tauopathies.

Another aspect of the present invention includes a method of promotingclearance of aggregates from the brain of a subject. This methodincludes administering a tau protein, its immunogenic epitopes, orantibodies recognizing the tau protein or its immunogenic epitopes underconditions effective to promote clearance of aggregates from the brainof a subject. The aggregates to be cleared include neurofibrillarytangles or their pathological tau precursors. Neurofibrillary tanglesare often associated with neurodegenerative diseases including, forexample, Alzheimer's disease, hereditary frontotemporal dementia andparkinsonism linked to chromosome 17 (FTDP-17), Pick's disease, sporadiccorticobasal degeneration, and progressive supranuclear palsy.

Another aspect of the present invention includes a method of slowing theprogression of a tangle-related behavioral phenotype in a subject. Thismethod includes administering a tau protein, its immunogenic epitopes,or antibodies recognizing the tau protein or its immunogenic epitopesunder conditions effective to slow a tangle-related behavioral phenotypein a subject.

The tau protein or immunogenic fragment described above can include anyone of the six isoforms of the human tau protein or a segment thereof.Tau has 0, 1, or 2 N-terminal inserts resulting from the splicing ofexons two and three, and either 3 or 4 microtubule-binding domainsresulting from the splicing of exon ten. The amino acid sequencescorresponding to the isoforms of the human tau protein of the presentinvention are given in SEQ ID NOs:21-26 below.

SEQ ID NO:21, the longest tau isoform (441 a.a), containing twoN-terminal inserts and four microtubule binding (2N4R) domains is asfollows: Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15 Thr Tyr GlyLeu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30 Gln Asp Gln GluGly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Len        35          40                          45 Gln Thr Pro Thr GluAsp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60 Asp Ala Lys Ser Thr ProThr Ala Glu Asp Val Thr Ala Pro Len Val65                  70                  75                  80 Asp GluGly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu                85                  90                  95 Ile Pro GluGly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro            100                 105                 110 Ser Leu Glu AspGlu Ala Ala Gly His Val Thr Gln Ala Arg Met Val        115                 120                 125 Ser Lys Ser Lys AspGly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly    130                 135                 140 Ala Asp Gly Lys Thr LysIle Ala Thr Pro Arg Gly Ala Ala Pro Pro145                 150                 155                 160 Gly GlnLys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                165                 170                 175 Pro Ala ProLys Thr Pro Pro Ser Ser Gly Gin Pro Pro Lys Ser Gly            180                 185                 190 Asp Arg Ser GlyTyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser        195                 200                 205 Arg Ser Arg Thr ProSer Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys    210                 215                 220 Lys Val Ala Val Val ArgThr Pro Pro Lys Ser Pro Ser Ser Ala Lys225                 230                 235                 240 Ser ArgLeu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val                245                 250                 255 Lys Ser LysIle Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly            260                 265                 270 Gly Lys Val GlnIle Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln        275                 280                 285 Ser Lys Cys Gly SerLys Asp Asn Ile Lys His Val Pro Gly Gly Gly    290                 295                 300 Ser Val Gln Ile Val TyrLys Pro Val Asp Leu Ser Lys Val Thr Ser305                 310                 315                 320 Lys CysGly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln            325                     330                 335 Val Glu ValLys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser            340                 345                 350 Lys Ile Gly SerLeu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn        355                 360                 365 Lys Lys Ile Glu ThrHis Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala    370                 375                 380 Lys Thr Asp His Gly AlaGlu Ile Val Tyr Lys Ser Pro Val Val Ser385                 390                 395                 400 Gly AspThr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                405                 410                 415 Ile Asp MetVal Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val            420                 425                 430 Ser Ala Ser LeuAla Lys Gln Gly Leu         435                 440

SEQ ID NO:22 contains two N-terminal inserts and threemicrotubule-binding domains (2N3R) as follows: Met Ala Glu Pro Arg GlnGlu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15 Thr Tyr GlyLeu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30 Gln Asp Gln GluGly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu        35                  40                  45 Gln Thr Pro Thr GluAsp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60 Asp Ala Lys Ser Thr ProThr Ala Glu Asp Val Thr Ala Pro Leu Val65                  70                  75                  80 Asp GluGly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu                85                  90                  95 Ile Pro GluGly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro            100                 105                 110 Ser Len Glu AspGlu Ala Ala Gly His Val Thr Gln Ala Arg Met Val        115                 120                 125 Ser Lys Ser Lys AspGly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly    130                 135                 140 Ala Asp Gly Lys Thr LysIle Ala Thr Pro Arg Gly Ala Ala Pro Pro145                 150                 155                 160 Gly GlnLys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                165                 170                 175 Pro Ala ProLys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly            180                 185                 190 Asp Arg Ser GlyTyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser        195                 200                 205 Arg Ser Arg Thr ProSer Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys    210                 215                 220 Lys Val Ala Val Val ArgThr Pro Pro Lys Ser Pro Ser Ser Ala Lys225                 230                 235                 240 Ser ArgLeu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val                245                 250                 255 Lys Ser LysIle Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly            260                 265                 270 Gly Lys Val GlnIle Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr        275                 280                 285 Ser Lys Cys Gly SerLeu Gly Asn ILe His His Lys Pro Gly Gly Gly    290                 295                 300 Gln Val Glu Val Lys SerGlu Lys Leu Asp Phe Lys Asp Arg Val Gln305                 310                 315                 320 Ser LysIle Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly                325                 330                 335 Asn Lys LysIle Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys            340                 345                 350 Ala Lys Thr AspHis Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val        355                 360                 365 Ser Gly Asp Thr SerPro Arg His Leu Ser Asn Val Ser Ser Thr Gly    370                 375                 380 Ser Ile Asp Met Val AspSer Pro Gln Leu Ala Thr Leu Ala Asp Glu385                 390                 395                 400 Val SerAla Ser Leu Ala Lys Gln Gly Leu                 405                 410

SEQ ID NO:23 contains one N-terminal insert and four microtubule-bindingdomains (IN4R) as follows: Met Ala Glu Pro Arg Gln Glu Phe Glu Val MetGlu Asp His Ala Gly1               5                   10                  15 Thr Tyr GlyLeu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30 Gln Asp Gln GluGly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu        35                  40                  45 Gln Thr Pro Thr GluAsp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60 Asp Ala Lys Ser Thr ProThr Ala Glu Ala Glu Glu Ala Gly Ile Gly65                  70                  75                  80 Asp ThrPro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala                85                  90                  95 Arg Met ValSer Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys            100                 105                 110 Ala Lys Gly AlaAsp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala        115                 120                 125 Ala Pro Pro Gly GlnLys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala    130                 135                 140 Lys Thr Pro Pro Ala ProLys Thr Pro Pro Ser Ser Gly Glu Pro Pro145                 150                 155                 160 Lys SerGly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr                165                 170                 175 Pro Gly SerArg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg            180                 185                 190 Glu Pro Lys LysVal Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser        195                 200                 205 Ser Ala Lys Ser ArgLeu Gln Thr Ala Pro Val Pro Met Pro Asp Leu    210                 215                 220 Lys Asn Val Lys Ser LysIle Gly Ser Thr Glu Asn Leu Lys His Gln225                 230                 235                 240 Pro GlyGly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser                245                 250                 255 Asn Val GlnSer Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro            260                 265                 270 Gly Gly Gly SerVal Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys        275                 280                 285 Val Thr Ser Lys CysGly Ser Leu Gly Asn Ile His His Lys Pro Gly    290                 295                 300 Gly Gly Gln Val Glu ValLys Ser Glu Lys Leu Asp Phe Lys Asp Arg305                 310                 315                 320 Val GlnSer Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly                325                 330                 335 Gly Gly AsnLys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn            340                 345                 350 Ala Lys Ala LysThr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro        355                 360                 365 Val Val Ser Gly AspThr Ser Pro Arg His Leu Ser Asn Val Ser Ser    370                 375                 380 Thr Gly Ser Ile Asp MetVal Asp Ser Pro Gln Leu Ala Thr Leu Ala385                 390                 395                 400 Asp GluVal Ser Ala Ser Leu Ala Lys Gln Gly Leu                405                 410

SEQ ID NO:24 contains zero N-terminal inserts and fourmicrotubule-binding domains (0N4R) as follows: Met Ala Glu Pro Arg GlnGlu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15 Thr Tyr GlyLeu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30 Gln Asp Gln GluGly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala        35                  40                  45 Gly Ile Gly Asp ThrPro Ser Leu Glu Asp Glu Ala Ala Gly His Val    50                  55                  60 Thr Gln Ala Arg Met ValSer Lys Ser Lys Asp Gly Thr Gly Ser Asp65                  70                  75                  80 Asp LysLys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro                85                  90                  95 Arg Gly AlaAla Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg            100                 105                 110 Ile Pro Ala LysThr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly        115                 120                 125 Glu Pro Pro Lys SerGly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser    130                 135                 140 Pro Gly Thr Pro Gly SerArg Ser Arg Thr Pro Ser Leu Pro Thr Pro145                 150                 155                 160 Pro ThrArg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys                165                 170                 175 Ser Pro SerSer Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met            180                 185                 190 Pro Asp Leu LysAsn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu        195                 200                 205 Lys His Gln Pro GlyGly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu    210                 215                 220 Asp Leu Ser Asn Val GlnSer Lys Cys Gly Ser Lys Asp Asn Ile Lys225                 230                 235                 240 His ValPro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp                245                 250                 255 Leu Ser LysVal Thr Ser LyS Cys Gly Ser Leu Gly Asn Ile His His            260                 265                 270 Lys Pro Gly GlyGly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe        275                 280                 285 Lys Asp Arg Val GlnSer Lys Ile Gly Ser Leu Asp Asn Ile Thr His    290                 295                 300 Val Pro Gly Gly Gly AsnLys Lys Ile Glu Thr His Lys Leu Thr Phe305                 310                 315                 320 Arg GluAsn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr                325                 330                 335 Lys Ser ProVal Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn            340                 345                 350 Val Ser Ser ThrGly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala        355                 360                 365 Thr Leu Ala Asp GluVal Ser Ala Ser Leu Ala Lys Gln Gly Leu    370                 375                 380

SEQ ID NO:25 contains one N-terminal insert and threemicrotubule-binding domains (1N3R) as follows: Met Ala Glu Pro Arg GlnGlu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15 Thr Tyr GlyLeu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30 Gln Asp Gln GluGly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu        35                  25                  45 Gln Thr Pro Thr GluAsp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60 Asp Ala Lys Ser Thr ProThr Ala Glu Ala Glu Glu Ala Gly Ile Gly65                  70                  75                  80 Asp ThrPro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala                85                  90                  95 Arg Met ValSer Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys            100                 105                 110 Ala Lys Gly AlaAsp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala        115                 120                 125 Ala Pro Pro Gly GlnLys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala    130                 135                 140 Lys Thr Pro Pro Ala ProLys Thr Pro Pro Ser Ser Gly Glu Pro Pro145                 150                 155                 160 Lys SerGly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr                165                 170                 175 Pro Gly SerArg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg            180                 185                 190 Glu Pro Lys LysVal Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser        195                 200                 205 Ser Ala Lys Ser ArgLeu Gln Thr Ala Pro Val Pro Met Pro Asp Leu    210                 215                 220 Lys Asn Val Lys Ser LysIle Gly Ser Thr Glu Asn Leu Lys His Gln225                 230                 235                 240 Pro GlyGly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser                245                 250                 255 Lys Val ThrSer Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro            260                 265                 270 Gly Gly Gly GlnVal Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp        275                 280                 285 Arg Val Gln Ser LysIle Gly Ser Leu Asp Asn Ile Thr His Val Pro    290                 295                 300 Gly Gly Gly Asn Lys LysIle Glu Thr His Lys Leu Thr Phe Arg Glu305                 310                 315                 320 Asn AlaLys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser                325                 330                 335 Pro Val ValSer Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser            340                 345                 350 Ser Thr Gly SerIle Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu        355                 360                 365 Ala Asp Glu Val SerAla Ser Leu Ala Lys Gln Gly Leu    370                 375                 380

SEQ ID NO.26 contains zero N-terminal inserts and threemicrotubule-binding domains (0N3R) as follows: His Met Ala Met Ala GluPro Arg Gln Glu Phe Glu Val Met Glu Asp1               5                   10                  15 His Ala GlyThr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr            20                  25                  30 Thr Met His GlnAsp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala        35                  40                  45 Glu Glu Ala Gly IleGly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala    50                  55                  60 Gly His Val Thr Gln AlaArg Met Val Ser Lys Ser Lys Asp Gly Thr65                  70                  75                  80 Gly SerAsp Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile                85                  90                  95 Ala Thr ProArg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn            100                 105                 110 Ala Thr Arg IlePro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro        115                 120                 125 Ser Ser Gly Glu ProPro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser    130                 135                 140 Pro Gly Ser Pro Gly ThrPro Gly Ser Arg Ser Arg Thr Pro Ser Leu145                 150                 155                 160 Pro ThrPro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr                165                 170                 175 Pro Pro LysSer Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro            180                 185                 190 Val Pro Met ProAsp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr        195                 200                 205 Glu Asn Leu Lys HisGln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr    210                 215                 220 Lys Pro Val Asp Leu SerLys Val Thr Ser Lys Cys Gly Ser Leu Gly225                 230                 235                 240 Asn IleHis His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu                245                 250                 255 Lys Leu AspPhe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp            260                 265                 270 Asn Ile Thr HisVal Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His        275                 280                 285 Lys Leu Thr Phe ArgGln Asn Ala Lys Ala Lys Thr Asp His Gly Ala    290                 295                 300 Glu ILe Val Tyr Lys SerPro Val Val Ser Gly Asp Thr Ser Pro Arg305                 310                 315                 320 His LeuSer Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser                325                 330                 335 Pro Gln LeuAla Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys            340                 345                 350 Gln Gly Leu        355

The tau protein of the present invention can be phosphorylated at one ormore amino acid residues. In a preferred embodiment, the tau protein isfully phosphorylated. Amino acid residues in the full length tauprotein, SEQ ID NO:21, that are phosphorylated include tyrosines atamino acid positions 18, 29, 97, 310, and 394; serines at amino acidpositions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293,324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonines at aminoacids positions 175, 181, 205, 212, 217, 231, and 403. Phosphorylatedamino acid residues in SEQ ID NO:22 include tyrosines at positions 18,29, 197, 279, and 363; serines at positions 184, 185, 198, 199, 202,208, 214, 235, 237, 238, 262, 293, 325, 365, 369, 373, 378, 381, 382,391; and threonine at positions 175, 181, 205, 212, 217, 231, 372.Phosphorylated amino acid residues in SEQ ID NO:23 include tyrosines atpositions 18, 29, 168, 281, and 365; serines at positions 155, 156, 169,170, 173, 179, 185, 206, 208, 209, 233, 264, 295, 327, 367, 371, 375,380, 383, 384, 393; and threonines at positions 146, 152, 176, 183, 188,202, and 374. Phosphorylated amino acids in SEQ ID NO:24 includetyrosines at positions 18, 29, 139, 252, 336; serines at positions 126,127, 140, 141, 144, 150, 156, 177, 179, 180,204, 235, 266, 298, 338,342, 346, 351, 354, 355, 364, and threonines at positions 117, 123, 147,154, 159, 173, and 345. Phosphorylated amino acid residues in SEQ ID NO:25 include tyrosines at positions 18, 29, 168, 250, 334; serines atpositions 155, 156, 169, 170, 173, 179, 185, 206, 208, 209, 233, 264,296, 336, 340, 344, 349, 352, 353, 362; and threonines at positions 146,152, 1376, 183, 188, 202, 343. Phosphorylated amino acid residues in SEQID NO: 26 include tyrosines at positions 18, 29, 139, 221, and 305;serines at positions 126, 127, 140, 141, 144, 150, 156, 177, 179,180,204, 235, 267, 307, 311, 315, 320, 323, 324, 333; and threonine atpositions 117, 123, 147, 154, 159, 173, and 314. Additional tyrosine,serine or threonine amino acids within the tau sequences may also bephosphorylated.

Unless otherwise indicated, reference to tau includes the natural humanamino acid sequences (SEQ ID NO:21-26). Variants of such segments,analogs, and mimetics of the natural tau peptide that induce and/orcrossreact with antibodies to the preferred epitopes of tau protein canalso be used. Analogs, including allelic, species, and induced variants,typically differ from naturally occurring peptides at one, two, or a fewpositions, often by virtue of conservative substitutions. Analogstypically exhibit at least 80 or 90% sequence identity with naturalpeptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids at one, two, or a fewpositions.

In addition to wildtype or natural tau proteins, the use of tau proteinscontaining one or more amino acid substitutions are also contemplated.In a preferred embodiment of the present invention, the tau proteincontains a proline to leucine mutation at amino acid position 301(P301L) of SEQ ID NO:21. Other amino acid mutations of the tan proteinare also contemplated. These mutations include a lysine to threoninemutation at amino acid residue 257 (K257T) in SEQ ID NO: 21; anisoleucine to valine mutation at amino acid position 260 (1260V) of SEQID NO:21; a glycine to valine mutation at amino acid position 272(G272V) of SEQ ID NO:21; an asparagine to lysine mutation at amino acidposition 279 (N279K) of SEQ ID NO:21; an asparagine to histidinemutation at amino acid position 296 (N296H) of SEQ ID NO:21; a prolineto serine mutation at amino acid position 301 (P301S) of SEQ ID NO;21; aglycine to valine mutation at amino acid position 303 (G303V) of SEQ IDNO:21; a serine to asparagine mutation at position 305 (S305N) of SEQ IDNO:21; a glycine to serine mutation at amino acid position 335 (G335S)of SEQ ID NO:21; a valine to methionine mutation at position 337 (V337M)of SEQ ID NO:21; a glutamic acid to valine mutation at position 342(E342V) of SEQ ID NO:21; a lysine to isoleucine mutation at amino acidposition 369 (K3691) of SEQ ID NO:21; a glycine to arginine mutation atamino acid position 389 (G389R) of SEQ ID NO:21; and an arginine totryptophan mutation at amino acid position 406 (R406W) of SEQ ID NO:21.In a preferred embodiment of the present invention, the tau mutantprotein or peptide fragment is phosphorylated.

Immunogenic fragments of the tau protein useful for the presentinvention can be identified based on sequence antigenicity,hydrophilicity, and accessibility. In a preferred embodiment, the tauprotein or its immunogenic epitopes may be phosphorylated at one or moreamino acids. One preferred immunogenic epitope of the tau protein ofthis invention is Tau379-408 containing phosphorylated serine residuesat positions 396 and 404. The sequence for Tau 379-408[P-Ser_(396,404)](SEQ ID NO:2) is shown in Table I as follows: SEQ ID NO: NAME SEQUENCESEQ ID NO:1 Tau 133-162 DGTGSDDKKAKGADGKTKIATPRGAAPPGQ-NH₂ SEQ ID NO:2Tau 379-409 [P-Ser_(396,404)]

SEQ ID NO:3 Tau 192-221 [P-Ser_(199,202,214,)-Thr_(205,212])

SEQ ID NO:4 Tau221-250 [P-Thr_(231,)-Ser₂₃₅]

SEQ ID NO:5 Tau184-213 SSGEPPKSGDRSQYSSPGSPGTPGSRSRT-NH₂ SEQ ID NO:6Tau1-30

SEQ ID NO:7 Tau30-60 TMHQDQEGDTDAGLKESPLQTPTEDGSEEPG-NH₂ SEQ ID NO:8Tau60-90 GSETSDAKSTPTAEDVTAPLVDEGAPGKQAA-NH₂ SEQ ID NO:9 Tau90-120AAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG-NH₂ SEQ ID NO:10 Tau120-150GHVTQARMVSKSKDGTGSDDKKAKGADGKTK-NH₂ SEQ ID NO:11 Tau150-180

SEQ ID NO:12 Tau180-210

SEQ ID NO:13 Tau210-240

SEQ ID NO:14 Tau240-270

SEQ ID NO:15 Tau270-300

SEQ ID NO:16 Tau300-330

SEQ ID NO:17 Tau330-360

SEQ ID NO:18 Tau360-390 ITHVPGGGNKKIETHKLTFRENAKAKTDHGA-NH₂ SEQ ID NO:19Tau390-420

SEQ ID NO:20 Tau411-441

Additional immunogenic fragments of the present invention include anyone SEQ ID NOs:1 or 3-20 as shown in Table 1. The names of the peptidesin Table 1 correspond to the amino acid position of these peptides inthe longest isoform of tau, SEQ ID NO:21. Many of these sequencescontain well established phospho-tau epitopes previously observed in ADand transgenic mouse models. Phosphorylated amino acid residues withineach sequence in Table 2 are indicated in bold and shading. TheC-terminus of each of SEQ ID NOs:1-20 above is preferably amidated asshown to preserve the immunogenicity of that region of the peptide.

In a preferred embodiment of the present invention, the tau peptides ofthe present invention can contain one or more D-amino acid residues. Theamino acids being in U-form would have the effect of enhancing thestability of the peptide. These D-amino acids can be in the same orderas the L-form of the peptide or assembled in a reverse order from theL-form sequence to maintain the overall topology of the native sequence(Ben-Yedidia et al., “A Retro-Inverso Peptide Analogue of InfluenzaVirus Hemagglutinin B-cell Epitope 91-108 Induces a Strong Mucosal andSystemic Immune Response and Confers Protection in Mice after IntranasalImmunization,” Mol Immunol. 39:323 (2002); Guichard, et al., “AntigenicMimicry of Natural L-peptides with Retro-Inverso-Peptidomimetics,” PNAS91:9765-9769 (1994); Benkirane, et al., “Antigenicity and Immunogenicityof Modified Synthetic Peptides Containing D-Amino Acid Residues,” J.Bio. Chem. 268(35):26279-26285 (1993), which are hereby incorporated byreference in their entirety).

Therapeutic agents can be longer polypeptides that include, for example,an active fragment of tau peptide, together with other amino acids. Forexample, preferred agents include fusion proteins comprising a segmentof tau linked to a promiscuous T-helper cell epitope which therebypromotes a B-cell response against the tau segment.

Other portions of the tau protein that are suitable for practicing thepresent invention include recombinant forms of the protein and fragmentsof the protein involved in the formation of paired helical filament(PHF). For example, PHF generated from tau, purified PHF from human ADbrains or purified PHF-like protein from P301L mice are additionalimmunogenic peptides contemplated for use when practicing the presentinvention.

Immunogenic fragments that have a low β-sheet content and few T-cellepitopes are preferred, however, fragments that contain high β-sheetcontent or T-cell epitopes can be modified to reduce potential toxicity.

Tau, its fragments, and analogs can be synthesized by solid phasepeptide synthesis or recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems (FosterCity, Calif.). Recombinant expression systems can include bacteria, suchas E. coli, yeast, insect cells, or mammalian cells. Procedures forrecombinant expression are described by Sambrook et al., MolecularCloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989), which ishereby incorporated by reference in its entirety.

In a variation of the present invention, an immunogenic peptide, such asa fragment of tau, can be presented by a virus or bacteria as part of animmunogenic composition. A nucleic acid encoding the immunogenic peptideis incorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of bacteria so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable.

Immune responses against neurofibrillary tangles can also be induced byadministration of nucleic acids encoding segments of tau peptide, andfragments thereof other peptide immunogens, or antibodies and theircomponent chains used for passive immunization. Such nucleic acids canbe DNA or RNA. A nucleic acid segment encoding an immunogen is typicallylinked to regulatory elements, such as a promoter and enhancer, whichallow expression of the DNA segment in the intended target cells of apatient. For expression in blood cells, as is desirable for induction ofan immune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie et al., Cur. Opin. Genet. Develop. 3:102-109(1993), which is hereby incorporated by reference in its entirety);adenoviral vectors (Bett et al., J. Virol. 67:5911 (1993), which ishereby incorporated by reference in its entirety); adeno-associatedvirus vectors (Zhou et al., J. Exp. Med. 179:1867 (1994), which ishereby incorporated by reference in its entirety), viral vectors fromthe pox family including vaccinia virus and the avian pox viruses, viralvectors from the alpha virus genus, such as those derived from Sindbisand Semliki Forest Viruses (Dubensky et al., J. Virol 70:508-519 (1996),which is hereby incorporated by reference in its entirety), Venezuelanequine encephalitis virus (see U.S. Pat. No. 5,643,576 to Johnston etal., which is hereby incorporated by reference in its entirety) andrhabdoviruses, such as vesicular stomatitis virus (see WO 96/34625 toRose, which is hereby incorporated by reference in its entirety) andpapillomaviruses (Ohe, et al., Human Gene Therapy 6:325-333 (1995); WO94/12629 to Woo et al.; and Xiao & Brandsma, Nucleic Acids. Res.24:2630-2622 (1996), which are hereby incorporated by reference in theirentirety).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. No. 5,208,036 to Eppstein et al., U.S. Pat. No.5,264,618 to Felgner et al., U.S. Pat. No. 5,279,833 to Rose, and U.S.Pat. No. 5,283,185 to Epand et al., which are hereby incorporated byreference in their entirety. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346 to Anderson etal., which is hereby incorporated by reference in its entirety). Suchvectors can further include facilitating agents such as bupivacine (U.S.Pat. No. 5,593,970 to Attardo et al., which is hereby incorporated byreference in its entirety). DNA can also be administered using a genegun (Xiao & Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996), which ishereby incorporated by reference in its entirety). The DNA encoding animmunogen is precipitated onto the surface of microscopic metal beads.The microprojectiles are accelerated with a shock wave or expandinghelium gas, and penetrate tissues to a depth of several cell layers. Forexample, the Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see WO 95/05853 to Carson et al.,which is hereby incorporated by reference in its entirety).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

Antibodies that specifically bind to any of the six isoforms of the tauprotein or fragment thereof, hyperphosphorylated tau, aggregated tau, orany region of the paired helical filaments may be therapeuticallyeffective in the context of the present invention. Preferred antibodiesare those which recognize the phosphorylated form of the tau proteinisoforms set forth in SEQ ID NOs: 21-26 or the phosphorylatedimmunogenic fragments set forth in SEQ ID NOs: 1-20. Such antibodies canbe monoclonal or polyclonal, full-length, single-chain antibodies, ornanobodies. Antibodies may be non-human, chimeric, or humanizedantibodies. Preferred antibodies may bind specifically to the aggregatedform of tau without binding to the dissociated form. Alternatively, anantibody may bind specifically to the dissociated form without bindingto the aggregated form. An antibody may recognize other forms of tauthat accumulates in AD brain and related disorders. These forms differfrom the normal tau in terms of post-translational modification,glycation, proteolytic truncation, and racemization. Antibodies used intherapeutic methods usually have an intact constant region or at least asufficient portion of the constant region to interact with an Fcreceptor. Human isotype IgG1 is preferred because of it having thehighest affinity of human isotypes for the FcR1 receptor on phagocyticcells. Bispecific Fab fragments can also be used, in which one arm ofthe antibody has specificity for tau, and the other for an Fc receptor.Some antibodies bind to tau with a binding affinity greater than orequal to about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of tau. However, polyclonalsera can be specific to a particular segment of tau, such as tau379-408.Monoclonal antibodies bind to a specific epitope within tau that can bea conformational or nonconformational epitope.

In some methods, multiple monoclonal antibodies having bindingspecificities to different epitopes are used. Such antibodies can beadministered sequentially or simultaneously. Antibodies toneurofibrillary tangle components other than tau can also be used.

Administration of the tau protein, its immunogenic epitope, or anantibody recognizing the protein or epitope can be used as a therapy totreat Alzheimer's disease, or other tauopathy associated with thedevelopment of neurofibrillary tangles. Additionally, the administrationof the tau protein, its immunogenic epitope or antibody recognizing theprotein or epitope can also be used as a prophylactic treatment toprevent the onset of Alzheimer's disease, or other tauopathy associatedwith the neurofibrillary tangle.

Another aspect of the present invention relates to a pharmaceuticalcomposition containing (one or more of) the immunogenic epitopes of thetau protein. In addition to the immunogenic epitope, the pharmaceuticalcomposition also contains a pharmaceutical carrier and/or a suitableadjuvant as described below.

A further aspect of the invention relates to a phosphorylated tauprotein and a pharmaceutical composition containing the phosphorylatedtau protein. The phosphorylated tau protein can be the full-lengthprotein, an isoform, fragment, or a recombinant form of the protein.Likewise the phosphorylated tau protein can also contain one or moreamino acid mutations. In addition to the phosphorylated tau protein, thepharmaceutical composition also contains a pharmaceutical carrier and/ora suitable adjuvant as described below.

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease. Therefore, the presentmethods can be administered prophylactically to the general populationwithout the need for any assessment of the risk of the subject patient.The present methods are especially useful for individuals who do have aknown genetic risk of Alzheimer's disease. Such individuals includethose having relatives who have experienced his disease, and those whoserisk is determined by analysis of genetic or biochemical markers.Genetic markers of risk toward Alzheimer's disease include mutations inthe APP gene, particularly mutations at position 717 and positions 670and 671 referred to as the Hardy and Swedish mutations respectively.Other markers of risk are mutations in the presenilin genes, PS1 andPS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia by the presenceof risk factors described above. In addition, a number of diagnostictests are available for identifying individuals who have AD. Theseinclude measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by Alzheimer's Diseaseand Related Disorders Association criteria.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60, or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent over time. If the response falls, a booster dosage is indicated.In the case of potential Down's syndrome patients, treatment can beginantenatally by administering therapeutic agent to the mother or shortlyafter birth.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presented duringdevelopment of the disease. In therapeutic applications, compositions ormedicants are administered to a patient suspected of, or alreadysuffering from, such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Insome methods, administration of agent reduces or eliminates mildcognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. The amount of immunogendepends on whether adjuvant is also administered, with higher dosagesbeing required in the absence of adjuvant. The amount of an immunogenfor administration sometimes varies from 1-500 μg per patient and moreusually from 5-500 μg per injection for human administration.Occasionally, a higher dose of 1-2 mg per injection is used. Typicallyabout 10, 20, 50, or 100 μg is used for each human injection. The massof immunogen also depends on the mass ratio of immunogenic epitopewithin the immunogen to the mass of immunogen as a whole. Typically,10⁻³ to 10⁻⁵ micromoles of immunogenic epitope are used for eachmicrogram of immunogen. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. On any given day thata dosage of immunogen is given, the dosage is greater than 1 μg/patientand usually greater than 10 μg/patient if adjuvant is also administered,and greater than 10 μg/patient and usually greater than 100 μg/patientin the absence of adjuvant. A typical regimen consists of animmunization followed by booster injections at time intervals, such as 6week intervals. Another regimen consists of an immunization followed bybooster injections 1, 2, and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 1O mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per every two weeks or once a month or onceevery 3 to 6 months. In some methods, two or more monoclonal antibodieswith different binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly, oryearly. In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of 1-1000 μg/ml and in some methods 25-300 μg/ml.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal, or intramuscular means forprophylactic and/or therapeutic treatment. The most typical route ofadministration of an immunogenic agent is subcutaneous although otherroutes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. Intramuscular injectionon intravenous infusion are preferred for administration of antibody. Insome methods, particular therapeutic antibodies are injected directlyinto the cranium. In some methods, antibodies are administered as asustained release composition or device, such as a Medipad™ device (ElanPharm. Technologies, Dublin, Ireland).

Another aspect of the present invention is a combination therapy whereina tau protein, its immunogenic epitope, or antibodies recognizing thetau protein or immunogenic epitope is administered in combination withother agents that are effective for treatment of relatedneurodegenerative diseases. In the case of amyloidogenic diseases suchas, Alzheimer's disease and Down's syndrome, immune modulation to clearamyloid-beta (Aβ) deposits is an emerging therapy. Immunotherapiestargeting Aβ have consistently resulted in cognitive improvements. It islikely that tau and Aβ pathologies are synergistic. Therefore, acombination therapy targeting the clearance of both pathologies at thesame time may be more effective than targeting each individually. In thecase of Parkinson's Disease and related neurodegenerative diseases,immune modulation to clear aggregated forms of the α-synuclein proteinis also an emerging therapy. A combination therapy which targets theclearance of both tau and α-synuclein proteins simultaneously may bemore effective than targeting each individually.

Immunogenic agents of the present invention, such as peptides, aresometimes administered in combination with an adjuvant. A variety ofadjuvants can be used in combination with a peptide, such as tau, toelicit an immune response. Preferred adjuvants augment the intrinsicresponse to an immunogen without causing conformational changes in theimmunogen that affect the qualitative form of the response.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, and aluminum sulfate. Suchadjuvants can be used with or without other specific immunostimulatingagents, such as 3 De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP,polymeric or monomeric amino acids, such as polyglutamic acid orpolylysine. Such adjuvants can be used with or without other specificimmunostimulating agents, such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837 to VanNest et al., which is hereby incorporated by reference in its entirety),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfuidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-bloeked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (e) Ribi™ adjuvant system (RAS),(Ribi InunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80,and one or more bacterial cell wall components from the group consistingof monophosphoryllipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), preferably MPL+CWS (Detox™). Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Other adjuvants include cytokines, such as interleukins(IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),and tumor necrosis factor (TNF).

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with, or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel, indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. However, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirety) alone or optionally all combinations thereof are suitable forhuman administration.

Agents of the present invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980), which is hereby incorporated by reference in its entirety. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the present invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water, oil, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin. Peanut oil, soybean oil, and mineral oil are allexamples of useful materials. In general, glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Agents of the invention,particularly, antibodies, can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained release of the active ingredient. Anexemplary composition comprises monoclonal antibody at 5 mg/mL,formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mMNaCl, adjusted to pH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles, such as polylactide, polyglycolide, or copolymer, forenhanced adjuvant effect (Langer, et al., Science 249:1527 (1990);Hanes, et al., Advanced Drug Delivery Reviews 28:97-119 (1997), whichare hereby incorporated by reference in their entirety).

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdernal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391:851(1998), which is hereby incorporated by reference in its entirety).Co-administration can be achieved by using the components as a mixtureor as linked molecules obtained by chemical crosslinking or expressionas a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25:3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368:201-15 (1998), which are herebyincorporated by reference in their entirety).

EXAMPLES Example 1 Peptides

The peptide immunogen, Tau 379-408 [P-Ser_(396,404)] was synthesized atthe Keck facility (Yale University), by the solid-phase technique on ap-methylbenzhydrylamine resin, using a Biosearch SAM 2 synthesizer(Biosearch, Inc., San Rafael, Calif.). The peptide was cleaved from theresin with HF and then extracted with ether and acetic acid beforelyophilization. Subsequently, it was purified by HPLC with the use of areverse-phase support medium (Delta-Bondapak) on a 0.78×30 cm columnwith a 0-66% linear gradient of acetonitrile in 0.1% TFA.

Example 2 Expression and Purification of Recombinant Tau Protein

pcDNA3.1(+) mutant tau P301L construct, expressing tau which has twoN-terminal exons (58 residues) and contains all the microtubule (MT)binding repeats in the longest human tau isoform. The cDNA was subclonedin pET30a vector using standard methods. The plasmid DNA was preparedusing the Qiagen protocol (Qiagen, Chatsworth, Calif.), and used totransform Escherichia coli BL21-DE3 competent cells. The clones weresequenced after transformation to verify the presence of the desiredmutation and to confirm that the rest of the tau sequence was identicalwith the published human cDNA sequence.

Tau purification was performed as described (Connell et al., “Effects ofFTDP-17 Mutations on the In Vitro Phosphorylation of Tau by GlycogenSynthase Kinase 3Beta Identified by Mass Spectrometry DemonstrateCertain Mutations Exert Long-Range Conformational Changes,” FEBS Lett.493:40-44 (2001), which is hereby incorporated by reference in itsentirety). Transformed bacteria were grown in LB broth containingkanamycin. The expression plasmid within the transformed cells was theninduced to express the tau protein of interest by adding IPTG. Thebacterial suspension was then centrifuged and the pellets resuspended in50 mM MES buffer pH 6.5, with protease inhibitors (Complete, Roche,Indianapolis, Ind.), sonicated, and centrifuged at 15,000×g at 4° C. for25 min. The supernatant was added to equal volume of 0.5 M NaCl toseparate out tau in its soluble form. This solution was boiled until awhite precipitate formed, subsequently centrifuged at 4° C. for 2 h at100,000×g, and then tau was precipitated out of the supernatant withammonium sulphate. Following centrifugation at 4° C. for 30 min at15,000×g, he supernatant was removed and the tau pellets resuspended in50 mM MES, pH 6.5, containing 1 m DITT and 50 mM NaCl. Followingdialysis, the tau was purified with FPLC.

Example 3 Antibody Purification and FITC Labeling

A polystyrene mini column (Pierce, Rockford, Ill.) was loaded with 1 mlof Gamma bind plus sepharose matrix (Amersbam Biosciences, UK) andallowed to settle. The column was washed extensively (at least 5 bedvolumes) with binding buffer (0.01 M sodium phosphate, 0.15 M NaCl, and0.01 M EDTA, pH 7.0) to return the pH to the neutral range. The columnwas then incubated with 1 ml of phosphate buffered saline (PBS) dilutedmouse plasma for 5 min at room temperature. This was then re-appliedonce more over the column to increase the yield of bound antibodies.Unbound protein was washed from the column with the binding buffer.Elution buffer (250 μl, 0.5 M acetic acid, pH 3.0) was appliedsequentially to the column and aliquots of elution fractions werecollected. The samples were quantified using the BCA protein assay kit(Pierce) and were also resolved on a 10% SDS-PACE gel. The appropriatefractions containing the cleanest separation of IgG from other serumproteins (typically fractions 5-8) were pooled and dialyzed over nightat 4° C. in 0.1 M Tris buffer pH 7.4 to quickly neutralize the elutionreagent's pH, before using the samples for further experiments.

For the labeling studies, fluorescein isothiocyanate (FITC; Sigma) wascovalently conjugated to the IgG via primary amines. Briefly, 1 mg ofIgG was dissolved in 100 mM sodium carbonate-bicarbonate buffer, pH 9.0,to a final reaction volume of 0.25 ml, and reacted with 5× molar ratioof FITC by adding 50 μl of FITC (1 mg/ml) very slowly in 5 μl aliquots.The FITC was solubilized in anhydrous dimethyl sulfoxide (DMSO). Thereaction vial was then covered with aluminum foil, and incubated for 8 hat 4° C. with gentle stirring. Ammonium chloride was then added to 5 mMand the reaction was incubated at 4° C. for 2 h. Lastly, xylene cyanolwas added to 0.1%, and to separate unbound FITC, the mixture was appliedto a PD-10 (Sephadex G-25M; Amersham) column gel bed that had beenequilibrated with PBS solution. Subsequently, the flow through wascollected and the column was eluted with 2.5 ml of PBS, in 0.25 mlfractions. Two bands were visible during elution, and the conjugate waspresent in the first band (fractions 3-5), as determined by measuringthe absorbance of each fraction at 280 nm and then at 495 nm (The, etal., “Conjugation of Fluorescein Isothiocyanate to Antibodies. II. AReproducible Method,” Immunology 18:875-881 (1970), which is herebyincorporated by reference in its entirety). The IgG was purified from amouse with a high titer against the immunogen (Phos-tau) and purifiedmouse IgG (Sigma) from pooled mouse plasma was used in controls.

Example 4 Fibrillogenicity

Aliquots of the tau peptide immunogen prepared in 0.1 M Tris, pH 7.4were incubated for different times, and their fibril formation comparedto that of Aβ1-42. In vitro fibrillogenesis was evaluated by an assaybased on the fluorescence emission by thioflavin T (ThT), as previouslydescribed in Sigurdsson, et al., “Immunization with aNon-Toxic/Non-Fibrillar Amyloid-β Homologous Peptide Reduces Alzheimer'sDisease Associated Pathology in Transgenic Mice,” Am J Pathol159:439-447 (2001); Sigurdsson, et al., “An Attenuated Immune Responseis Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse ModelImmunized with Amyloid-beta Derivatives,” J Neurosci. 24:6277-6282(2004), which are hereby incorporated by reference in their entirety.ThT binds specifically to β-sheet structure and this binding produces ashift in its emission spectrum and a fluorescent enhancementproportional to the amount of amyloid formed. Following the incubationperiod, 50 mM glycine, pH 9.2, and 2 μM ThT was added to a final volumeof 200 μl containing 3 μg of the immunogen or the P301L tau peptide(with or without equimolar concentration of antibodies purified from theimmunized mice). Fluorescence was measured at excitation 435 nm andemission 485 nm on a SpectraMax M2 multi-detection plate reader(Molecular Devices, Sunnyvale, Calif.).

Example 5 Neurotoxicity

Potential neurotoxicity of the tau derivative (10 μM) (SEQ ID NO:2) wasevaluated at 6 days in a human neuroblastoma cell line (SK-N-SH) usingthe standard MTT assay as described by the manufacturer (Roche,Pleasanton, Calif.). AβI1-40 and Aβ1-42 were used as control peptides.

Example 6b Animals Used in Studies

The studies were performed in the transgenic (Tg) P301L mouse model thatdevelops neurofibrillary tangles in several brain regions and spinalcord (Taconic, Germantown, N.Y.) (Lewis, et al., “NeurofibrillaryTangles, Amyotrophy and Progressive Motor Disturbance in Mice ExpressingMutant (P301L) Tau Protein,” Nat Genet. 25:402-405 (2000), which ishereby incorporated by reference in its entirety). While this model isnot ideal for AD, it is an excellent model to study the consequences oftangle development and for screening therapy that may prevent thegeneration of these aggregates. Another advantage of these animals isthe relatively early onset of pathology. In the homozygous line,behavioral abnormalities associated with tau pathology can be observedat least as early as 3 months, but the animals remain relatively healthyat least until 8 months of age. In other words, at 8 months, the animalsambulate, feed themselves, and can perform the behavioral taskssufficiently well to allow the treatment effect to be monitored.

Example 7 Vaccine Administration

Phos-tau peptide was mixed with Adju-Phos adjuvant (Brenntag Biosector,Denmark) at a concentration of 1 mg/ml and the solution was rotatedovernight at 4° C. prior to administration to allow the peptide toadsorb onto the aluminum phosphate particles. The mice received asubcutaneous injection of 100 μl followed by a second injection 2 weekslater and then monthly thereafter. In the first study, vaccinationstarted at 2 months of age and continued until the animals were 5 monthsof age at which time the animals were perfused and their organscollected for analysis. In the latter study, the mice were immunizedstarting at 2 months of age and continued until the animals were 8months. The mice went through a battery of sensorimotor tests at 5months and again at 8 months of age prior to sacrifice. Control micereceived the adjuvant alone.

Example 8 Intracarotid Injection of FITC-Labeled Antibodies

Mice were anesthetized with 2% isoflurane and maintained with 1.5%isoflurane in 70% N₂O and 30% O₂. After exposing the carotid sheath,left common carotid artery (CCA), external carotid artery (ECA), andinternal carotid artery (ICA) were exposed via a midline incision. Asilk suture was tied to the distal end of the ECA, and the left CCA,ICA, and pterygopalatine artery (PPA) were temporarily tied. A 30 Gneedle connected to PE-10 tubing (Becton Dickinson, San Diego, Calif.)was attached to a 1 ml syringe, mounted on a pump, and the FITC-IgGconjugate was then administered over a period of 10-15 min through theneedle punctured upward into the common carotid. Subsequently, theneedle was slowly withdrawn and glue was applied to the site ofinjection to prevent postoperative bleeding. One hour later, the micewere perfused transaortically with PBS andperiodate-lysine-paraformaldehyde (PLP) and postfixed in PLP overnight.The brains were then placed overnight in a phosphate buffer solutioncontaining 20% glycerol and 2% DMSO. Subsequently, serial coronal 40 μmsections of the brain were prepared and subject to fluorescentmicroscopy.

Example 9 Antibody Response

The mice were bled prior to the commencement of the study and a weekfollowing each injection. The antibody response to the vaccine wasdetermined by serial dilution of plasma using an ELISA assay asdescribed previously (Sigurdsson, et al., “Immunization with aNon-Toxic/Non-Fibrillar Amyloid-β Homologous Peptide Reduces Alzheimer'sDisease Associated Pathology in Transgenic Mice,” Am J Pathot159:439-447 (2001); Sigurdsson, et al., toxic “An Attenuated ImmuneResponse is Sufficient to Enhance Cognition in an Alzheimer's DiseaseMouse Model Immunized with Amyloid-beta Derivatives,” J Neurosci.24:6277-6282 (2004), which are hereby incorporated by reference in theirentirety), where the immunogen was coated onto Immulon™ microtiter wells(Thermo Fischer Scientific, Waltham, Mass.,). For detection, goatanti-mouse IgG linked to a horseradish peroxidase (Amersham, Piscataway,N.J.) was used at 1:3000 dilution. Tetramethyl benzidine (Pierce) wasthe substrate.

Example 10 Histology

Mice were anesthetized with sodium pentobarbital (120 mg/kg, i.p.),perfused transaortically with PBS and the brains processed as previouslydescribed (Sigurdsson, et al., “Immunization with aNon-Toxic/Non-Fibrillar Amyloid-β Homologous Peptide Reduces Alzheimer'sDisease Associated Pathology in Transgenic Mice,” Am J Pathol.159:439-447 (2001); Sigurdsson, et al., “An Attenuated Immune Responseis Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse ModelImmunized with Amyloid-beta Derivatives,” J Neurosci. 24:6277-6282(2004); Sigurdsson E., “Histological Staining of Amyloid-beta in MouseBrains,” Methods Mol Biol. 299:299-308 (2005), which are herebyincorporated by reference in their entirety). Briefly, the righthemisphere was immersion fixed overnight inperiodate-lysine-paraformaldehyde (PLP), whereas the left hemisphere wassnap-frozen for tau protein analysis (see Western Blots below).Following fixation, the brain was moved to a phosphate buffer solutioncontaining 20% glycerol and 2% dimethylsulfoxide (DMSO) and stored at 4°C. until sectioned. Serial coronal brain sections (40 μm) were cut andevery tenth section was stained with two tau antibodies that recognizeabnormal tau protein (PHF1, MC1). The series were placed in ethyleneglycol cryoprotectant and stored at −20° C. until used.

Tau antibody staining was performed as described in Sigurdsson, et al.,“Immunization with a Non-Toxic/Non-Fibrillar Amyloid-β HomologousPeptide Reduces Alzheimer's Disease Associated Pathology in TransgenlcMice,” Am J Pathol 159:439-447 (2001); Sigurdsson, et al., “AnAttenuated Immune Response is Sufficient to Enhance Cognition in anAlzheimer's Disease Mouse Model Immunized with Amyloid-betaDerivatives,” J Neurosci. 24:6277-6282 (2004), which are herebyincorporated by reference in their entirety. Briefly, sections wereincubated in the tau primary antibodies, MC1 and PHF1, at a 1:50 to1:100 dilution. A mouse on mouse immunodetection kit (VectorLaboratories, Burlingame, Calif.) was used, in which the anti-mouse IgGsecondary antibody was used at a 1:2000 dilution.

Mice that received intracarotid injection of FITC-labeled purified mouseIgG were perfused one hour following the injection with PBS until theperfusate was clear and subsequently with PLP for 10 minutes followed byfurther PLP fixation overnight. The brain was then placed overnight inthe glycerol/DMSO phosphate buffer and then immediately sectioned,mounted, and coverslipped using fluorescence compatible mounting media(Dako). Adjacent sections were stained free-floating with PHF1 or MC1anti-tau antibodies followed by reaction with Texas red labeledanti-mouse IgG. Sections were counterstained with4′-6-diamidino-2-phenylindole (DAPI) that labels nuclei. In contrast tothe regular immunohistochemistry described above, blocking solution wasnot used prior to applying the primary antibody.

Example 11 Image Analysis

Analysis of tissue sections was quantified with a Bioquant imageanalysis system. The software uses hue, saturation, and intensity tosegment objects in the image field. Thresholds were established withaccurately identified objects on a standard set of slides and thesesegmentation thresholds remained constant throughout the analysissession. After establishing the threshold parameter, the image field wasdigitized with a frame grabber. The Bioquant software corrects forheterogeneity in background illumination (blank field correction) andcalculates the measurement parameter for the entire field. Forquantitative image analysis of immunohistochemistry, the granular layerof the dentate gyrus was initially selected which consistently containedintraneuronal tau aggregates (pretangles and tangles). This observationconcurs with the original characterization of this model (Lewis, et al.,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet. 25:402-405(2000), which is hereby incorporated by reference in its entirety). Themotor cortex and brain stem in these animals were also analyzed, becausetau pathology in those regions may relate to the motor abnormalitiesobserved in this model. All procedures were performed by an individualblind to the experimental conditions of the study. Sample numbers wererandomized before the start of the tissue processing, and the code wasbroken only after the analysis was complete. For each antibody stain(PHF1, MC1), every tenth section from the mouse brain was sampled. Inthe dentate gyrus, the measurement was the percent of area in themeasurement field at ×200 magnification occupied by reaction productwith the tip of the dentate gyrus at the left edge of the field. Four tofive sections were analyzed per animal. In the motor cortex, themeasurement was the percent of neuronal staining in the field at ×100magnification with the thickest region of the cingulum positioned at thelower left edge of the field. In the brain stem, the measurement was thepercent of neuronal staining in the field at ×100 magnification with thecenter of the top edge of the field positioned below the Aqueduct ofSylvius. Five sections were analyzed per animal in those two brainregions.

Example 12 Western Blotting

Brains were homogenized in Tris-buffered saline (TBS; 10 mM Tris/150 mMNaCl (H 7.4)) containing protease inhibitors (1 tablet in 10 ml TBS;Complete mini protease inhibitor cocktail tablet; Roche) and phosphataseinhibitors (1 mM NaF, 0.4 μM Na₃VO₄, and 0.5 μM okadaic acid). Afterinitial preclearance centrifugation at 20,800×g for 5 min, the postnuclear supernatants were centrifuged at 100,000×g for 60 min, and thesupernatants were collected. The resulting pellets were resuspended withan equal volume of homogenization buffer containing 0.1% SDS (pH 8.0) togenerate high speed soluble samples.

Equal amounts of protein (BCA assay) were loaded and the samples wereelectrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulosemembranes. All blots were blocked (5% nonfat milk and 0.1% Tween-20 inTBS), then incubated with various antibodies over night. Subsequently,the blots were washed and incubated for 1 h at room temperature withperoxidase-conjugated, goat anti-rabbit or anti-mouse IgG (1:2000,Amersham), followed with washing and detection of bound antibodies (ECL;Pierce). Immunoreactivity of tau proteins was analyzed from scannedfilms using U-SCAN-IT software. To compare the relative amount of tauprotein, the densities of the immunoreactive bands corresponding tophospho-tau were normalized and reported, relative to the amounts oftotal tau protein.

Example 13 Antibodies for Tau Histology and Blots

PHF1 mAb was used at 1:500 for immunoblots and 1:50 forimmunohistochemistry. PHF1 mab recognizes phosphorylated serines 396 and404 located within the microtubule-binding repeat on the C-terminal ofPHF tau protein (Otvos, et al., “Monoclonal Antibody PHF-1 RecognizesTau Protein Phosphorylated at Serine Residues 396 and 404,” J NeurosciRes, 39:669-673 (1994), which is hereby incorporated by reference in itsentirety). MC1 tau antibody was used as well (Jicha, et al., “SequenceRequirements for Formation of Conformational Variants of Tau Similar tothose found in Alzheimer's Disease,” J Neurosci Res. 55:713-723 (1999),which is hereby incorporated by reference in its entirety). Both theseantibodies recognize the neurofibrillary tangles in the P301L mousemodel (Lewis, et al., “Neurofibrillary Tangles, Amyotrophy andProgressive Motor Disturbance in Mice Expressing Mutant (P301L) TauProtein,” Nat Genet. 25:402-405 (2000), which is hereby incorporated byreference in its entirety). MC1 is a conformation dependent IgG1antibody that is similar to Alz-50 (IgM). Its reactivity depends on boththe NH₂ terminus (amino acids 7-9), and an amino acid sequence of tau(amino acids 313-322) in the third microtubule binding domain that isboth necessary and sufficient for in vitro formation of filamentousaggregates of tau similar to those seen in AD (Jicha, et al., “SequenceRequirements for Formation of Conformational Variants of Tau Similar tothose found in Alzheimer's Disease,” J Neurosci Res. 55:713-723 (1999),which is hereby incorporated by reference in its entirety). Thepathological conformation of tau recognized by MC1 may precede theaggregation of tau into filaments and the resultant neurofibrillarydegeneration seen in AD (Jicha, et al., “Sequence Requirements forFormation of Conformational Variants of Tau Similar to those found inAlzheimer's Disease,” J Neurosci Res. 55:713-723 (1999), which is herebyincorporated by reference in its entirety). 3G6 monoclonal antibody thatrecognizes total tau and anti-actin polyclonal antibody (1:2000; Sigma)were used as controls on Western blots.

Example 14 Behavioral Studies

Several tests were performed in the mice to determine; 1) If thetau-targeting therapy prevented or reversed the age-related sensorimotorabnormalities observed in the P301L mice; and 2) If the immunization hadany behavioral effects per se. Cognitive analysis of these homozygousanimals has not been previously reported, and it was determined thattheir sensorimotor defects would not enable them to properly navigatethe radial arm maze, which has been used extensively to assess thecognitive status of Tg2576 mice that deposit Aβ within the brain(Sigurdsson, et al., “An Attenuated Immune Response is Sufficient toEnhance Cognition in an Alzheimer's Disease Mouse Model Immunized withAmyloid-beta Derivatives,” J Neurosci. 24:6277-6282 (2004); Asuni, etal., “Vaccination of Alzheimer's Model Mice with Abeta Derivative inAlum Adjuvant Reduces Abeta Burden without Microhemorrhages,” Eur JNeurosci. 24: 2530-2542 (2006), which are hereby incorporated byreference in their entirety). Hence, an object recognition test thatrequires less navigation was used. The tests utilized include. 1)locomotor activity; 2) motor and reflex behaviors: a) traverse beamtest, b) accelerating rotarod; and 3) memory test: object recognition.Prior to sensorimotor testing, the mice were adapted to the room withlights on for 15 min.

Example 15 Locomotor Activity

Computerized recording of the activity of the animals over a designatedperiod of time was performed. Exploratory locomotor activity wasrecorded in a circular open field activity chamber (70 cm in diameter).A video camera mounted above the chamber automatically recordedhorizontal movements in the open field in each dimension (i.e., x, y,and two z planes). Total distance was measured in centimeters (cm)traveled and is defined as sequential movement interruptions of theanimal (white) measured relative to the background (black). The durationof the behavior was timed for 15 min. Results are reported based ondistance traveled (cm), mean resting time, and velocity (mean andmaximum) of the animal.

Example 16 Rotarod

The animals were placed onto the rod (diameter 3.6 cm) apparatus toassess differences in motor coordination and balance by measuring fore-and hindlimb motor coordination and balance (Rotarod 7650 acceleratingmodel; Ugo Basile, Biological Research Apparatus, Varese, Italy). Thisprocedure was designed to assess motor behavior without a practiceconfound. The animals were habituated to the apparatus by receivingtraining sessions of two trials, sufficient to reach a baseline level ofperformance. Then, the mice were tested three additional times, withincreasing speed. During habituation, the rotarod was set at 1.0 rpm,which was gradually raised every 30 sec, and was also wiped clean with30% ethanol solution after each session. A soft foam cushion was placedbeneath the apparatus to prevent potential injury from falling. Eachanimal was tested for three sessions, with each session separated by 15min, and measures were taken for latency to fall or invert (by clinging)from the top of the rotating barrel.

Example 17 Traverse Beam

This task tests balance and general motor coordination and functionintegration. Mice were assessed by measuring their ability to traverse agraded narrow wooden beam to reach a goal box (Torres, et al.,“Behavioural, Histochemical and Biochemical Consequences of SelectiveImmunolesions in Discrete Regions of the Basal Forebrain CholinergicSystem,” Neuroscience 63:95-122 (1994), which is hereby incorporated byreference in its entirety). The mice were placed on a 1.1 cm wide beamthat is 50.8 cm long and suspended 30 cm above a padded surface by twoidentical columns. Attached at each end of the beam is a shaded goalbox. Mice were placed on the beam in a perpendicular orientation tohabituate and were then monitored for a maximum of 60 sec. The number offoot slips each mouse had before falling or reaching the goal box wererecorded for each of four successive trials. Errors are defined asfootslips and were recorded numerically. To prevent injury from falling,a soft foam cushion was always kept underneath the beam. Animals thatfell off were placed back in their position prior to the fall.

Example 18 Object Recognition

The spontaneous object recognition test that was utilized measuresdeficits in short term memory, and was conducted in a square-shapedopen-field box (48 cm square, with 18 cm high walls constructed fromblack Plexiglas), raised 50 cm from the floor. The light intensity wasset to 30 lx. On the day before the tests, mice were individuallyhabituated in a session in which they were allowed to explore the emptybox for 15 min. During training sessions, two novel objects were placedat diagonal corners in the open field and the animal was allowed toexplore for 15 min. For any given trial, the objects in a pair were 10cm high, and composed of the same material so that they could notreadily be distinguished by olfactory cues. The time spent exploringeach object was recorded by a tracking system (San Diego Instruments,San Diego, Calif.), and at the end of the training phase, the mouse wasremoved from the box for the duration of the retention delay (RD=3 h).Normal mice remember a specific object after a delay of up to 1 h andspend the majority of their time investigating the novel object duringthe retention trial. During retention tests, the animals were placedback into the same box, in which one of the previous familiar objectsused during training was replaced by a second novel object, and allowedto explore freely for 6 min. A different object pair was used for eachtrial for a given animal, and the order of exposure to object pairs aswell as the designated sample and novel objects for each pair werecounterbalanced within and across groups. The time spent exploring thenovel and familiar objects was recorded for the 6 min. The percentageShort Term Memory score is the time spent exploring any one of the twoobjects (training session) compared to the novel one (retentionsession).

The objective of these experiments was to evaluate the effects of thevaccination on selected sensorimotor and cognitive behaviors. Thehomozygous P301L mice have tangle pathology as early as 3 months of ageand those animals were tested at 5- and 8 months of age.

Example 19 Data Analysis

All the data was analyzed with GraphPad Prism 4.3. The amount of tauaggregates on western blots, the immunuoreactivity on brain sectionswithin the dentate gyrus, motor cortex and brainstem, the locomotoractivity measurements (distance, Vmax, Vmean, rest time) and the objectrecognition test were analyzed by an unpaired t-test. When the datafailed at least two out of three normality tests (KS-, D'Agostino &Pearson omnibus-, and Shapiro-Wilk normality tests) non-parametricMann-Whitney test was used. The test was two-tailed when genderdifferences were analyzed but otherwise one-tailed because it waspredicted that the immunotherapy would clear tau pathology that wouldslow the progression of behavioral impairments. Neurotoxicity wasanalyzed by one-way ANOVA and Neuman Keuls post hoc test. The data fromthe traverse beam and rotarod were analyzed by two-way ANOVA repeatedmeasures and a Bonferroni post hoc test. Correlation between behavioraloutcome and tau pathology, as assessed by immunohistochemistry orWestern blotting, was analyzed by Pearson r correlation or Spearman rankcorrelation if the data failed normality test.

Example 20 In Vitro Assays

To determine the therapeutic potential of tau-based immunotherapy, aphosphorylated immunogen, Tau379-408[P-Ser_(396,404)](SEQ ID NO:2), wasdesigned that would lead to the generation of antibodies which wouldselectively detect highly phosphorylated tau protein as found in AD andtangle mouse models. The tau protein, like the Aβ peptide, has thepropensity to form P-sheets which have been associated with toxicity. Toexamine potential in vivo toxicity of the immunogen, itsfibrillogenicity and neurotoxicity were examined in vitro. Data from theThioflavin T assay demonstrate the immunogen is not fibrillogenic (FIG.1A). Likewise, data from the MTT assay indicate the immunogen is notneurotoxic to SK-N-SH neurons as compared with neurons treated with AP1-42 for 6 days (10 μM, 20% reduction in neuronal viability, p<0.01).

Example 21 Animal Studies

Treatment from 2-5 Months. Homozygous Tg P301L mice were immunized from2 months of age with the Phos-tau peptide in aluminum adjuvant (n=12, 5males and 7 females). Control Tg P301L animals received adjuvant alone(n=14, 8 males, 6 females). The mice elicited a robust antibody responseagainst the immunogen, whereas minimal reactivity was observed incontrol mice (FIG. 1B). Surprisingly, autoantibodies that recognizedrecombinant tau (P301L and wild-type) were observe in controls andimmunized mice (FIG. 1C). In the original description of a hemizygousline of this model, the most extensive pathology was observed in thebrain stem and spinal cord (Lewis, et al., “Neurofibrillary Tangles,Amyotrophy and Progressive Motor Disturbance in Mice Expressing Mutant(P301L) Tau Protein,” Nat Genet. 25:402-405 (2000), which is herebyincorporated by reference in its entirety). The homozygous line that wasutilized also has extensive pathology in these regions but appears tohave more pathology in the hippocampal and cortical regions thanhemizygous animals which is as expected because of their homozygocity.Quantitative analysis of neuronal immunoreactivity in the dentate gyruswith two antibodies that selectively stain Alzheimer's tau proteinrevealed 74%—(p<0.01; FIG. 2A, FIG. 3A,B) and 52% (p<0.05, FIG. 2B, FIG.3C,D) reduction in MC1- and PHF1 immunoreactivity, respectively. MC1recognizes earlier stage of tau pathology than PHF1 (Jicha, et al.,“Sequence Requirements for Formation of Conformational Variants of TauSimilar to those found in Alzheimer's Disease,” J Neurosci Res.55:713-723 (1999), which is hereby incorporated by reference in itsentirety), and these intermediates should be more easily cleared thanhigher order aggregates which may explain the greater reduction in MC1-compared to PHF1 immunoreactivity. To confirm this finding in otherbrain regions that may better relate to the motor abnormalities in themodel, neuronal MC1 staining was quantified in the motor cortex andbrain stem, and immunoreactivity was reduced by 96% (p<0.0001, FIG. 2C,FIG. 3E,F) and 93% (p=0.01, FIG. 2D, FIG. 3G, H), respectively, comparedto control Tg mice. No significant correlation was observed betweenantibody levels against the immunogen, recombinant wild-type tau orP301L tau versus the immunohistochemical analysis. Wester blot analysisdid not show as robust treatment effect as observed withimmunohistochemistry (FIG. 2E). Densitometric analysis of the PHF1 blotsrevealed a strong trend for reduction in insoluble tau (28% reduction,p−0.09) and a significant increase in soluble tau (77% increase, p=0.01)in the immunized mice compared to control Tg mice. No significantchanges were observed on MC1 blots although there was a strong trend foran increase in soluble tau (167% increase, p=0.10). Further analysis ofthe ratio of soluble tau to insoluble tau indicated a significantincrease in the immunized group on PHF1 blots (89% increase, p=0.01),suggesting a mobilization of tau from its insoluble form to soluble formin these treated animals. Furthermore, a positive correlation wasobserved between antibody levels against the immunogen versus banddensity in PHF1 blots (insoluble tau: r=−0.60, p<0.01; ratio of solubleto insoluble tau, r=0.46, p<0.03).

Gender Differences. More extensive tau pathology in females than maleshas been described in a hemizygous line of the P301L model (Lewis, etal., “Neurofibrillary Tangles, Amyotrophy and Progressive MotorDisturbance in Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet.25:402-405 (2000), which is hereby incorporated by reference in itsentirety) and this was considered in group assignments of the homozygousline of the P301L mice utilized. When gender differences in taupathology as assessed by immunohistochemistry were compared, the femalecontrols had more tau pathology in all areas analyzed than male controlsbut no gender differences were observed between immunized mice (Table2). When the immunohistochemical data based on gender was reanalyzed, agreater treatment effect was observed among the females than males(Table 2). TABLE 2 Immunohistochemical Analysis: Treatment Effect withinGender and Gender Differences. Dentate Gyrus (MC1) Dentate Gyrus (PHF1)M F M F3 2-5 months Tg Control 8.03 ± 2.44 37.05 ± 6.37⁺⁺⁺  8.91 ± 3.6223.96 ± 1.39⁺⁺ Tg Phos-tau 6.94 ± 3.03  4.13 ± 1.26**** 13.72 ± 6.89 2.78 ± 2.15*** 2-8 months Tg Control 7.39 ± 3.40 30.21 ± 2.17⁺⁺⁺  9.46± 6.78 24.54 ± 5.19 Tg Phos-tau 3.00 ± 1.89 17.18 ± 4.19**/⁺⁺  7.32 ±5.67 15.08 ± 6.38* Motor Cortex (MC1) Brain Stem (MC1) M F M F 2-5months Tg Control 0.89 ± 0.33 2.74 ± 0.48⁺⁺ 0.38 ± 0.12 4.72 ± 1.89⁺⁺ TgPhos-tau 0.09 ± 0.07* 0.06 ± 0.03*** 0.12 ± 0.04* 0.19 ± 0.16** 2-8months Tg Control 2.09 ± 1.60 2.83 ± 1.54   1.88 ± 1.37 5.12 ± 1.29 TgPhos-tau 0.08 ± 0.07** 0.36 ± 0.12*  0.23 ± 0.18* 1.39 ± 0.31**/⁺⁺Treatment Effect within Gender: Greater effect of the immunization wasobserved between females (8 out of 8 groups) than males (4 out of 8groups).*, **, ***p ≦ 0.05, 0.01, 0.001. Significantly different from controlmice within the same gender.Gender Differences: Significant gender differences were observed betweensome of the groups. In the 2-5 months study, males (n = 8) and female (n= 6) controls differed in all the areas analyzed but not phos-tauimmunized males (n = 5) and females (n = 7). In the 2-8 months study (6males and 6 females per group),# this difference was not as group specific with differences observed inthe dentate gyrus (MC1) within both treatment groups and in the brainstem in the phos-tau immunized mice.⁺, ⁺⁺, ⁺⁺⁺p ≦ 0.05, 0.01, 0.001. Significantly different from maleswithin the same treatment group.

A similar gender-related pattern was observed on Western blots (Table3A), particularly in the ratio of soluble to insoluble tau (Table 3B),but gender specific treatment effect was not pronounced on the blots.TABLE 3A-B Western Blot Analysis Treatment Effect within Gender andGender Differences. A. Percentage density relative to total tau values.Insoluble Tau Soluble Tau Insoluble Tau Soluble Tau PHF1 PHF1 MC1 MC1 MF M F M F M F 2-5 months Tg Control 184 ± 35 96 ± 33 122 ± 15 169 ± 5712 ± 3 17 ± 3  7 ± 1  3 ± 1⁺ Tg Phos-tau 116 ± 12 95 ± 13 282 ± 46** 218± 52 18 ± 6 27 ± 8 20 ± 9  7 ± 2* 2-8 months Tg Control  39 ± 11 96 ± 24111 ± 15  55 ± 6⁺⁺ 11 ± 3 15 ± 6 54 ± 14 12 ± 4⁺⁺ Tg Phos-tau  38 ± 1368 ± 14  77 ± 14  62 ± 5 11 ± 2 32 ± 21 56 ± 13  9 ± 3⁺⁺ B. Ratio ofvalues in A (soluble/insoluble). Ratio Ratio (soluble/insoluble)(soluble/insoluble) PHF1 MC1 M F M F 2-5 months Tg Control 0.83 ± 0.142.04 ± 0.41⁺⁺ 0.76 ± 0.15 0.16 ± 0.04⁺⁺ Tg Phos-tau 2.72 ± 0.62** 2.36 ±0.50 1.55 ± 0.90 0.36 ± 0.11 2-8 months Tg Control 4.26 ± 1.19 1.00 ±0.38⁺ 5.73 ± 1.64 2.07 ± 0.88 Tg Phos-tau 2.98 ± 0.61 1.31 ± 0.45 5.20 ±1.39 0.83 ± 0.34⁺⁺Treatment Effect within Gender: (A) Greater effect of the immunizationwas observed between males in levels of soluble PHF1 tau and in femalesin levels of soluble MC1. In addition, the ratio of soluble/insolubletau differed between treated- and control mice in PHF1 blots for males.*, **: p ≦ 0.05, 0.01. Significantly different from control mice withinthe same gender.Gender Differences: Significant gender differences were observed betweensome of the groups but these differences were not as prevalent as in theimmunohistochemical analysis. In the 2-5 months study, males (n = 8) andfemale (0 = 6) controls differed in soluble tau levels (MC1) as well astau ratio (soluble/insoluble; PHF1 and MC1) but not# phos-tau immunized males (n = 5) and females (n = 7). A similarpattern was observed in the immunohistochemical analysis withdifferences only observed in control groups. # In the 2-8 months study(6 males and 6 females per group), this difference was not as groupspecific with differences observed in soluble tau (MC1, PHF1) as well astau ratio (soluble/insoluble; PHF1) within controls. In the phos-tauimmunized mice, gender differences were detected in soluble tau (MC1) aswell as tau ratio (soluble/insoluble; MC1).⁺, ⁺⁺; p ≦ 0.05, 0.01. Significantly different from males within thesame treatment group.

Treatment from 2-8 Months. Following these promising findings, alongitudinal study was performed in another set of 2 month oldhomozygous P301L mice (n=12 per group, 6 males and 6 females per group).Again, a robust antibody response was elicited against the Phos-tauderivative and some, although much less reactivity was observed incontrol mice (FIG. 4A). Again, the plasma obtained at the end of thestudy in both the controls and immunized mice contained autoantibodiesthat recognized recombinant tau (P301L and wild-type; FIG. 4B). In these8 month old animals, the level of these autoantibodies was substantiallyhigher than in the 5 month old mice in the previous study (see FIG. 1C).It is well known that autoantibodies increase with age and applicantshave observed a similar phenomenon in the Tg2576 mouse model of cerebralAβ amyloidosis, although those animals are usually well into theirsecond year when appreciable levels of autoantibodies are detected(Sigurdsson, et al., “An Attenuated Immune Response is Sufficient toEnhance Cognition in an Alzheimer's Disease Mouse Model Immunized withAmyloid-beta Derivatives,” J Neurosci. 24:6277-6282 (2004); Asuni, etal., “Vaccination of Alzheimer's Model Mice with Abeta Derivative inAlum Adjuvant Reduces Abeta Burden without Microhemorrhages,” Eur JNeurosci. 24: 2530-2542 (2006), which are hereby incorporated byreference in their entirety).

At 5 months of age, these animals underwent their first behavioraltesting to determine if the therapy was associated with functionalimprovements. The immunization increased the time the animals were ableto stay on the accelerating rotarod (p<0.02, FIG. 5A), and reduced thenumber of foot slips in the traverse beam task (p<0.001, FIG. 5B). Also,the vaccinated mice attained higher maximum velocity (Vmax: p=0.004,FIG. 5C) in the locomotor activity test but were not significantlydifferent from Tg controls in the distance traveled, average speed(Vmean), or the resting time. These animals continued to receive monthlyimmunizations and were retested on the same behavioral tests at 8 monthsof age. The mice were subsequently killed for biochemical andhistological analysis of tau pathology. At 8 months of age, the treatedanimals continued to perform better than Tg controls on the rotarod andtraverse beam although the differences between the groups were not assubstantial as during the earlier comparison (Rotarod: p<0.05, FIG. 5A;Traverse Beam: p=0.05, FIG. 5B). The locomotor activity of the groupswas similar at this time point (Vmax in FIG. 5C). Overall, the groupsperformed worse at 8 months compared to 5 months. Because of therelatively poor mobility of the animals at this age, cognitiveassessment was only performed with an object recognition test. This testindicated that both the immunized mice and their controls werecognitively normal with the animals spending 60-70% of their timeexploring the novel object (FIG. 5D), which is similar to wild-typemouse performance.

Quantitative analysis of tau immunoreactivity in the dentate gyrosrevealed a 47% reduction (p<0.05, FIG. 6A) in MC1 staining and a strongtrend (40% reduction, p=0.12, FIG. 6B) for reduced PHF1immunoreactivity. As at 5 months of age, MC1 neuronal staining was morerobustly reduced in the motor cortex (76%, p=0.02, FIG. 6C) and brainstem (78%, p=0.005, FIG. 6D) than in the dentate gyrus. Biochemicalanalysis of the whole left hemisphere of the brain did not revealsignificant differences in pelletable tau or soluble tau on PHF1 or MC1blots. Hence as expected, no correlation was observed between antibodylevels against the immunogen vs. band density on PHF1- or MC1 blots ofbrain homogenate.

Interestingly, performance on behavioral assays that require extensivemotor coordination (traverse beam and rotarod) correlated with taupathology in corresponding brain areas (motor cortex and brain stem;Table 4). TABLE 4 Behavioral assays at 8 months that correlatedsignificantly with subsequent immunohistochemical analysis. Motor CortexBrain Stem (MC1) (MC1) Traverse Beam r = 0.47 r = 0.47 (8 months) p =0.01 p = 0.01 Rotarod r = −0.37 r = −0.45 (8 months) p = 0.04 p = 0.02Of the behavioral assays performed at 5 and 8 months in the 2-8 monthsstudy, only the performance of the mice on the traverse beam and rotarodat 8 months showed any correlation with the subsequentimmunohistochemical analysis. Notably, the only correlation was foundbetween assays that require extensive motor coordination (traverse beamand rotarod) and corresponding brain areas (motor cortex and brainstem). r = Spearman r, p-value (one-tailed).In addition, antibody levels against the immunogen, but not therecombinant tau proteins (wild-type and P301L), at the time of sacrifice(T4) correlated with tau pathology in the brain stem (MC1, r=−0.48,p<0.01), and dentate gyrus (MC1, r=−0.37, p=0.04, PHF1, r=−0.44,p=0.02), and there was a trend for a similar correlation in the motorcortex (MC1, r=−0.23, p=0.14).

Gender Differences. As in the treatment study that lasted from 2-5months, significant gender differences in immunohistochemical taupathology were also observed in the 2-8 months study group. Again,females had more pathology than males although only in one brain regionin controls (MC1: dentate gyrus) and in two brain regions in immunizedmice (MC1: dentate gyrus and brain stem; Table 2). In contrast, in the2-5 months study group, the males and females in the control groupdiffered in all the brain regions analyzed but no gender differenceswere observed in the immunized mice (Table 2). As in the 2-5 monthgroup, greater treatment effect was observed among the females thanmales when the immunohistochemical data based on gender was reanalyzed(Table 2). A similar gender-related pattern was observed on Westernblots with the females having more insoluble tau and less soluble tauthan their male counterparts (Table 3). However, gender specifictreatment effect was not observed at this time point.

Interestingly, although these gender differences in tau pathology wereclearly observed, this pattern was not seen in the behavioral analysis.The only significant difference between males and females within thesame group was observed in the rotarod at 5 months (p<0.05), with thefemales performing better (3.6±0.2 rpm) than the males (2.8±0.1 rpm).

Example 22 Antibody Characterization

The high levels of autoantibodies observed in the animals complicatescharacterization of the antibodies that were generated towards theimmunogen. Purified antibodies from several of the immunized micestained specifically tau aggregates/tangles in neuronal cell bodies ofP301L mice similar to the PHF-antibody and did not react with tau inwild-type mice (FIG. 7). But a similar staining pattern could beobserved with antibodies purified from control animals with high levelsof tau autoantibodies although most of them resulted in minimal or nostaining in P3OIL or wild-type mice (FIG. 7). These findings indicatethat the immunized mice generate antibodies that specifically recognizepathological tau aggregates in the P301L mouse, but some animals maycontain autoantibodies with these properties. Likewise, the antibodiesfrom the immunized mice stained tangle pathology in Alzheimer's brain(FIG. 8). By staining selected mouse brain sections with secondaryantibody against mouse IgG, intracerebral antibodies that label neuronswere detected in the P301L mice (FIG. 9).

To confirm that anti-tau antibodies would gain access into the brain,purified IgG antibodies from an immunized mouse that had generated veryhigh levels of antibodies against the immunogen were FITC-tagged. TheFITC-labeled IgG was subsequently injected into the carotid artery of 8month old P301L mice and their brains were harvested one hour later foranalysis of antibody uptake. Several neurons in various brain regionsshowed typical green FITC fluorescence (FIG. 10), and, when the sectionswere incubated with PHF1 or MC1, these antibodies colocalized with theFITC-labeled IgG (FIG. 11). Carotid injection in another set of 8 monthold P301L mice of FITC-labeled purified IgG from a control mouse thatreceived only the alum adjuvant, resulted in some FITC fluorescencewithin the brain although it did not appear to be neuronal and it didnot colocalize with PHF1 or MC1 staining. The identical approach inwild-type mice with the same antibodies did not lead to FITCfluorescence within the brain, indicating that leakage of the bloodbrain barrier (BBB) in the P301L model may at least in part explaintreatment efficacy.

These results demonstrate that. 1) vaccination of P301L mice with aphospho-tau epitope leads to the generation of antibodies that enter thebrain; 2) these antibodies bind to abnormal tau like a monoclonalantibody (PHF1) against a similar epitope; 3) this type of immunotherapyreduces the extent of aggregated tau in the brain and slows theprogression of the behavioral phenotype of these animals; and 4) asexpected, the therapeutic effect decreases as the functional impairmentsadvance in these animals.

Regarding the mechanism of tau-based immunotherapy, it is wellestablished that about 0. 1% of circulating IgG is found within the CNSerenberg, et al., “Radioimmunoassays for Ig Classes G, A, M, D, and E inSpinal Fluids; Normal Values of Different Age Groups,” J Lab Clin Med.86:887-898 (1975), which is hereby incorporated by reference in itsentirety), and it may enter through regions that are deficient in BBB(Broadwell, et al., “Serum Proteins Bypass the Blood-Brain FluidBarriers for Extracellular Entry to the Central Nervous System,” ExpNeurol. 120; 245-263 (1993), which is hereby incorporated by referencein its entirety), as has been shown for an antibody targeting Aβ (Banks,et al., “Passage of Amyloid Beta Protein Antibody across the Blood-BrainBarrier in a Mouse Model of Alzheimer's Disease,” Peptides 23:2223-2226(2002), which is hereby incorporated by reference in its entirety).Also, IgG can cross the BBB via adsorptive-mediated transcytosis(Zlokovic, et al., “A Saturable Mechanism for Transport ofImmunoglobulin G Across the Blood-Brain Barrier of the Guinea Pig,” ExpNeurol 107:263-270 (1990), which is hereby incorporated by reference inits entirety). The BBB is thought to be compromised in variousneurological disorders such as AD, suggesting that a substantiallygreater percentage of circulating IgG can be found within the CNS.Increased permeability of the BBB has been observed in plaque depositingAD model mice (Poduslo, et al., “Permeability of Proteins at theBlood-Brain Barrier in the Normal Adult Mouse and Double TransgenicMouse Model of Alzheimer's Disease,” Neurobiol Dis. 8:555-567 (2001);LaRue, et al., “Method for Measurement of the Blood-Brain BarrierPermeability in the Perfused Mouse Brain: Application to Amyloid-betaPeptide in Wild type and Alzheimer's Tg2576 Mice,” Journal ofNeuroscience Methods 138:233-242 (2004), which are hereby incorporatedby reference in their entirety), but has not been assessed in tangle ADmodel mice. However, the present observations indicate that the BBB islikely to be impaired in the P301L model. Besides antibody uptake intothe brain, it is also well established that antibody secreting cellsfrom the periphery can enter the brain and secrete the antibodieslocally (Knopf, et al., “Antigen-Dependent Intrathecal AntibodySynthesis in the Normal Rat Brain: Tissue Entry and Local Retention ofAntigen-Specific B Cells,” Journal of Immunology 161:692-701 (1998),which is hereby incorporated by reference in its entirety). In the mouseimmunotherapy studies, targeting Aβ, IgG has been routinely found withinthe brain associated with extracellular Aβ deposits and phagocyticmicroglia (Schenk D., “Amyloid-beta Immunotherapy for Alzheimer'sDisease: The End of the Beginning,” Nat Rev Neurosci. 3:824-828 (2002),which is hereby incorporated by reference in its entirety), and in thepresent study neuronal antibodies have been detected within the brain byimmunohistochemistry. In addition, FITC-labeled IgG was detected inbrains of P301IL mice but not in wild-type mice following intracarotidinjection which indicates that antibodies can enter the brain from theperiphery in the P301L model. It is interesting to note that the levelsof autoantibodies against tau increased with age, suggesting thatage-related impairments of the BBB associated with the progression ofbrain pathology in these animals may expose the tau proteins as anantigen to the immune system.

Transport of antibodies within the CNS has not been thoroughlyinvestigated, but IgG transport within and across cells in the peripheryis essential for effective humoral immunity. Also, several studies haveshown that antibodies can be found within neurons, which supports thefeasibility of the present approach (for example, see Fabian, et al.,“Intraneuronal IgG in the Central Nervous System,” J Neurol Sci.73:257-267 (1986); Fabian, et al., “Intraneuronal IgG in the CentralNervous System: Uptake by Retrograde Axonal Transport,” Neurology37:1780-1784 (1987); Liu, et al., “Immunohistochemical Localization ofIntracellular Plasma Proteins in the Human Central Nervous System,” ActaNeuropathol (Berl). 78; 16-21 (1989); Dietzschold, et al., “Delineationof Putative Mechanisms Involved in Antibody-Mediated Clearance of RabiesVirus from the Central Nervous System” Proc Natl Acad Sci USA89:7252-7256 (1992) (published erratum appears in Proc Natl Acad Sci USA89(19):9365 (1992)); Aihara, et al., “Immunocytochemical Localization ofImmunoglobulins in the Rat Brain: Relationship to the Blood-BrainBarrier,” J Comp Neurol. 342:481-496 (1994); Mohamed, et al.,“Immunoglobulin Fc Gamma Receptor Promotes Immunoglobulin Uptake,Immunoglobulin-Mediated Calcium Increase, and Neurotransmitter Releasein Motor Neurons,” J Neurosci Res. 69:110-116 (2002), which are herebyincorporated by reference in their entirety). The antibodies may enterthe cells via pinocytosis, e.g. receptor-mediated endocytosis orfluid-phase endocytosis (Lobo, et al., “Antibody Pharmacokinetics andPharmacodynamics,” J Pharm Sci. 93:2645-2668 (2004), which is herebyincorporated by reference in its entirety). In cells that do not havesurface antigens recognized by the antibody, receptor-mediated uptakemay occur via Fcγ or FcRn receptors (Lobo, et al., “AntibodyPharmacokinetics and Pharmacodynamics,” J Pharm Sci. 93:2645-2668(2004), which is hereby incorporated by reference in its entirety), andFcγ receptors have been located on neurons (Mohamed, et al.,“Immunoglobulin Fc Gamma Receptor Promotes Immunoglobulin Uptake,Immunoglobulin-Mediated Calcium Increase, and Neurotransmitter Releasein Motor Neurons,” J Neurosci Res. 69:110-116 (2002); Andoh, et al.,“Direct Action of Immunoglobulin G on Primary Sensory Neurons Through FcGamma Receptor I,” FASEB J. 18:182-184 (2004), which are herebyincorporated in their entirety by reference). Indeed, it has beendemonstrated that central neurons that project to the periphery, such asmotor neurons, take up IgG at their synapses by retrograde axonaltransport through receptors for the Fe portion of IgG (Fabian, et al.,“Intraneuronal IgG in the Central Nervous System: Uptake by RetrogradeAxonal Transport,” Neurology 37:1780-1784 (1987); Mohamed, et al.,“Immunoglobulin Fc Gamma Receptor Promotes Immunoglobulin Uptake,Immunoglobulin-Mediated Calcium Increase, and Neurotransmitter Releasein Motor Neurons,” J Neurosci Res, 69:110-116 (2002), which are herebyincorporated by reference in their entirety). One strong possibility isthat circulating anti-tau antibodies in the CNS may recognize andcross-link abnormally conformed neuronal plasma membrane associated tau.As a matter of fact, tau has been shown to be associated with neuralplasma membrane components in addition to microtubules, and there isevidence that this interaction is influenced by phosphorylation of tauat sites that are modified in paired helical filaments (PHFs) (Ditella,et al., “Microfilament-Associated Growth Cone Component Depends Upon Taufor Its Intracellular-Localization,” Cell Motility and the Cytoskeleton29:117-130 (1994); Brandt, et al., “Interaction of Tau with the NeuralPlasma-Membrane Mediated by Tau Amino-Terminal Projection Domain,” JCell Biol. 131:1327-1340 (1995); Ekinci, et al., “Phosphorylation of TauAlters its Association with the Plasma Membrane,” Cellular and MolecularNeurobiology 20:497-508 (2000); Maas, et al., “Interaction of Tau withthe Neural Membrane Cortex is Regulated by Phosphorylation at Sites thatare Modified in Paired Helical Filaments,” J Biol Chem. 275:15733-15740(2000), which are hereby incorporated by reference in their entirety).In addition, tau interacts with actin (Correas, et al., “TheTubulin-Binding Sequence of Brain Microtubule-Associated Proteins, Tauand Map-2, is also Involved in Actin Binding,” Biochemical Journal269:61-64 (1990), which is hereby incorporated by reference in itsentirety)) and spectrin (Carlier, et al., “Interaction BetweenMicrotubule-Associated Protein Tau and Spectrin,” Biochimie 66: 305-311(1984), which is hereby incorporated by reference in its entirety),which may provide another link to the neural membrane.

Clearance of extracellular tangles may reduce associated pathology, and,because of the numerous reports of cellular uptake of antibodies some ofwhich are cited above, intracellular tangles and pre-tangles may also becleared, which could prevent neuronal damage. Recent findings from arelated field support the validity of the present approach andsubsequent observations. Like tau in AD, α-synuclein aggregatesintracellularly within the brain in Parkinson's disease, andimmunization with α-synuclein in mice with this pathology clears theseaggregates (Masliah, et al., “Effects of Alpha-Synuclein Immunization ina Mouse Model of Parkinson's Disease,” Neuron 46:857-868 (2005), whichis hereby incorporated by reference in its entirety).

Less effort has been spent on developing therapy targeting pathologicaltau conformers than their Aβ counterparts. With regard to immunotherapy,Aβ plaque clearance in the AN 1792 trial did not appear to affect tanglepathology (Nicoll, et al., “Neuropathology of Human Alzheimer Diseaseafter Immunization with Amyloid-beta Peptide: A Case Report,” Nat Med.9:448-452 (2003); Ferrer, et al., “Neuropathology and Pathogenesis ofEncephalitis Following Amyloid-beta Immunization in Alzheimer'sDisease,” Brain Pathol. 14:11-20 (2004); Masliah, et al., “AbetaVaccination Effects on Plaque Pathology in the Absence of Encephalitisin Alzheimer Disease,” Neurology 64:129-131 (2005), which are herebyincorporated by reference in their entirety). However, Aβ immunotherapyin a mouse model cleared early tau pathology but not hyperphosphorylatedtau aggregates (Oddo, et al., “Abeta Immunotherapy Leads to Clearance ofEarly, but not Late, Hyperphosphorylated Tau Aggregates via theProteasome,” Neuron 43:321-332 (2004), which is hereby incorporated byreference in its entirety). These findings emphasize the need fortherapy targeting pathological tau conformers. In Aβ plaque mousemodels, it may not be necessary to clear plaques to observe a cognitivebenefit (Sigurdsson E., “Immunotherapy for Conformational Diseases,”Current Pharmaceutical Design 12:2569-2585 (2006), which is herebyincorporated by reference in its entirety). This concept may also applyto tau pathology, because suppression of transgenic tau in a tanglemouse model has been shown to improve memory although neurofibrillarytangles remained (Santacruz, et al., “Tau Suppression in aNeurodegenerative Mouse Model Improves Memory Function,” Science309:476-481 (2005), which is hereby incorporated by reference in itsentirety). In that same model, region-specific dissociation of neuronalloss and neurofibrillary pathology has been observed (Spires, et al.,“Region-Specific Dissociation of Neuronal Loss and NeurofibrillaryPathology in a Mouse Model of Tauopathy,” Am J Pathol. 168:1598-1607(2006), which is hereby incorporated by reference in its entirety) whichsuggests toxic effects of early stage pathological tau conformers thatcannot easily he detected, analogous to Aβ oligomers. A similar lack ofcorrelation of tan pathology with neuronal death was previously reportedin another tangle model, hTau, that overexpresses unmutated human tau ona mouse tau knockout background (Andorfer, et al., “Cell-Cycle Reentryand Cell Death in Transgenic Mice Expressing Nonmutant Human TauIsoforms,” J Neurosci. 25: 5446-5454 (2005), which is herebyincorporated by reference in its entirety). Considering thesediscrepancies, it is interesting that although the model employed herehas age-related progression in tau pathology and functional impairments,immunohistochemical analysis revealed a similar degree of tau pathologyat 5 and 8 months and the animals performed at a comparable level on theRotarod and the Traverse Beam at these different ages. However, theirlocomotor activity was substantially less at 8 months compared to 5months. It is also of note that a significant correlation was observedbetween performance on the Rotarod and the Traverse Beam and taupathology in two out of the three brain areas analyzed, namely the motorcortex and the brain stem (see Table 3) both of which have a prominentrole in motor coordination, which indicates a direct relationshipbetween the main pathological feature of this model and associatedfunctional impairments.

The immunotherapy approach used here was substantially more effective inthe early stages of functional impairments in the animals (at 5 months)than at a later time point (at 8 months). These findings indicate thatclearance of early stage pathological tau conformers may be of atherapeutic benefit. These smaller aggregates should also be easier toclear than late stage neurofibrillary tangles. As previously reported inthe original heterozygous line of this model (Lewis, et al.,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet. 25:402-405(2000), which is hereby incorporated by reference in its entirety), thehomozygous females had more extensive neurofibrillary pathology than themales as observed by immunohistochemistry (see Table 2). It was,therefore, surprising that the immunotherapy was more effective in thefemales. Similar gender differences in therapeutic outcome in othertransgenic models of tau pathology have not been reported. A possibleexplanation is that more of the aggregated tau in the females isavailable for antibody-mediated disassembly because of its higher levelswithin the neurons. In the male animals, the lower amount ofpathological tau may be more easily sequestered and thereby lessaccessible for antibody binding. A related possibility is that becauseof its higher levels and presumably more rapid assembly, the tauaggregates in the females may be more soluble and hence more easilyremoved. The Western blot data did not correlate exactly with theimmunohistochemical findings. However, similar gender differences weredetected and a more robust treatment effect was observed with both typesof analysis in the 5 month old group compared to the 8 month oldanimals. Any discrepancy may at least in part be related to theartificial antibody epitopes that are generated in the blots. It is wellestablished that both the MC1- and PHF1 antibodies show greaterspecificity towards pathological tau on histological sections than inWestern blots (Greenberg, et al., “Hydrofluoric Acid-Treated Tau PHIFProteins Display the Same Biochemical Properties as Normal Tau,” J BiolChem. 267:564-569 (1992); Rye, et al., “The Distribution of Alz-50Immunoreactivity in the Normal Human Brain,” Neuroscience 56:109-127(1993); Jicha, et al., “Alz-50 and MC-1, a New Monoclonal AntibodyRaised to Paired Helical Filaments, Recognize Conformational Epitopes onRecombinant Tau,” Journal of Neuroscience Research 48:128-132 (1997);Weaver, et al., “Conformational Change as one of the EarliestAlterations of Tau in Alzheimer's Disease,” Neurobiol Aging 21:719-727(2000), which are hereby incorporated by reference in their entirety).

It should be emphasized that these tau immunotherapy studies wereperformed in transgenic mice that are homozygous for the tau mutationP301L. These animals have a very aggressive phenotype with tau pathologydetected within a few months of age and with related severe functionalimpairments observed a few months later. The present approach should bemore effective in transgenic animals with a phenotype progression thatmore resembles the human condition, and eventually in humans withfrontotemporal dementia and/or AD in which pathology develops over yearsor decades instead of months.

Overall, the present findings support the feasibility of immunotherapytargeting pathological tau conformers that may benefit AD patients andindividuals with frontotemporal dementia caused by tau mutations.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of treating or preventing Alzheimer's Disease or othertauopathies in a subject, said method comprising: administering a tauprotein, its immunogenic epitopes, or antibodies recognizing the tauprotein or its immunogenic epitopes under conditions effective to treator prevent Alzheimer's Disease or other tauopathies.
 2. The methodaccording to claim 1, wherein the tau protein and its immunogenicepitopes are phosphorylated and the antibodies recognize phosphorylatedforms of the tau protein and its immunogenic epitopes.
 3. The methodaccording to claim 1, wherein a tau protein is administered.
 4. Themethod according to claim 3, wherein the tau protein is selected fromthe group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26.
 5. The method according to claim1, wherein an immunogenic epitope of the tau protein is administered. 6.The method according to claim 5, wherein the immunogenic epitope of thetau protein has an amino acid sequence selected from any one of SEQ IDNOs: 1-20.
 7. The method according to claim 6, wherein the immunogenicepitope of the tau protein has an amino acid sequence of SEQ ID NO:2. 8.The method according to claim 1, wherein an antibody recognizing the tauprotein or its immunogenic epitopes is administered.
 9. The methodaccording to claim 8, wherein the antibody is a monoclonal antibody, apolyclonal antibody, or binding portions thereof.
 10. The methodaccording to claim 1, wherein Alzheimer's Disease is treated.
 11. Themethod according to claim 15 wherein Alzheimer's Disease is prevented.12. A method of promoting clearance of aggregates from the brain of asubject, said method comprising: administering a tau protein, itsimmunogenic epitopes, or antibodies recognizing the tau protein or itsimmunogenic epitopes under conditions effective to promote clearance ofaggregates from the brain of a subject.
 13. The method according toclaim 12, wherein the tau protein and its immunogenic epitopes arephosphorylated and the antibodies recognize phosphorylated forms of thetau protein and its immunogenic epitopes.
 14. The method according toclaim 12, wherein a tau protein is administered.
 15. The methodaccording to claim 14, wherein the tau protein is selected from thegroup consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26.
 16. The method according to claim12, wherein an immunogenic epitope of the tau protein is administered.17. The method according to claim 16, wherein the immunogenic epitope ofthe tau protein has an amino acid sequence selected from any one of SEQID NOs: 1-20.
 18. The method according to claim 17, wherein theimmunogenic epitope of the tau protein has an amino acid sequence of SEQID NO:2.
 19. The method according to claim 12, wherein an antibodyrecognizing the tau protein or its immunogenic epitopes is administered.20. The method according to claim 19, wherein the antibody is amonoclonal antibody, a polyclonal antibody, or binding portions thereof.21. The method according to claim 12, wherein the aggregates areneurofibrillary tangles or their pathological tau precursors.
 22. Amethod of slowing progression of a tangle-related behavioral phenotypein a subject, said method comprising: administering a tau protein, itsimmunogenic epitopes, or antibodies recognizing the tau protein or itsimmunogenic epitopes under conditions effective to slow a tangle-relatedbehavioral phenotype in a subject.
 23. The method according to claim 22,wherein the tau protein and its immunogenic epitopes are phosphorylatedand the antibodies recognize phosphorylated forms of the tau protein andits immunogenic epitopes.
 24. The method according to claim 22, whereina tau protein is administered.
 25. The method according to claim 24,wherein the tau protein is selected from the group consisting of SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQID NO:26.
 26. The method according to claim 22, wherein an immunogenicepitope of the tau protein is administered.
 27. The method according toclaim 26, wherein the immunogenic epitope of the tau protein has anamino acid sequence selected from any one of SEQ ID NOs: 1-20.
 28. Themethod according to claim 27, wherein the immunogenic epitope of the tauprotein has an amino acid sequence of SEQ ID NO:2.
 29. The methodaccording to claim 22, wherein an antibody recognizing the tau proteinor its immunogenic epitopes is administered.
 30. The method according toclaim 29, wherein the antibody is a monoclonal antibody, a polyclonalantibody, or binding portions thereof.
 31. A peptide comprising animmunogenic epitope of a tau protein, wherein the peptide has an aminoacid sequence selected from any one of SEQ ID NOs:1-20.
 32. The peptideaccording to claim 31, wherein the peptide has the amino acid sequenceof SEQ ID NO:2.
 33. The peptide according to claim 31, wherein thepeptide is phosphorylated.
 34. The peptide according to claim 31,wherein the peptide is linked to a promiscuous t-helper cell epitope.35. A pharmaceutical composition comprising: the peptide according toclaim 31, a pharmaceutically acceptable adjuvant effective to induce animmune response, and a pharmaceutical carrier.
 36. An antibody orbinding portion thereof recognizing the peptide according to claim 31.37. The antibody or binding portion thereof according to claim 36,wherein the peptide is phosphorylated.
 38. The antibody or bindingportion thereof according to claim 36, wherein the antibody is amonoclonal antibody.
 39. The antibody or binding portion thereofaccording to claim 36, wherein the antibody is a polyclonal antibody.40. A combination immunotherapeutic comprising: the antibody accordingto claim 36 and an antibody or binding portion thereof recognizing theamyloid-β peptide or α-synuclein peptide.
 41. A phosphorylated tauprotein.
 42. A pharmaceutical composition comprising: the proteinaccording to claim 41 and a pharmaceutical carrier and/or an adjuvant.